The spatial self-organization within pluripotent stem cell colonies is continued in detaching aggregates.

Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotency. To investigate the dynamics of this process under spatial confinement, we used either polydimethylsiloxane (PDMS) pillars or micro-contact printing of vitronectin. There was a progressive upregulation of OCT4, E-cadherin, and NANOG within 70 µm from the outer rim of iPSC colonies. Single-cell RNA-sequencing and spatial reconstruction of gene expression demonstrated that OCT4high subsets, residing at the edge of the colony, have pronounced up-regulation of the TGF-ß pathway, particularly of NODAL and its inhibitor LEFTY. Interestingly, after 5-7 days, iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size without enzymatic or mechanical treatment. Within the early 3D aggregates, radial organization and differential gene expression continued in analogy to the changes observed during self-organization of iPSC colonies. Early self-detached aggregates revealed up-regulated germline-specific gene expression patterns as compared to conventional EBs. However, there were no marked differences after further directed differentiation toward hematopoietic, mesenchymal, and neuronal lineages. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs.

Electrochemical Impedance Spectroscopy Biosensor Enabling Kinetic Monitoring of Fucosyltransferase Activity.

Monitoring glycosyltransferases on biosensors is of great interest for pathogen and cancer diagnostics. As a proof of concept, we here demonstrate the layer-by-layer immobilization of a multivalent neoglycoprotein (NGP) as a substrate for a bacterial fucosyltransferase (FucT) and the subsequent binding of the fucose-specific Aleuria aurantia lectin (AAL) on an electrochemical impedance spectroscopy (EIS) sensor. We report for the first time the binding kinetics of a glycosyltransferase in real-time. Highly stable EIS measurements are obtained by the modification of counter and reference electrodes with polypyrrole: polystyrene sulfonate (PPy:PSS). In detail, the N-acetyllactosamine (LacNAc)-carrying NGP was covalently immobilized on the gold working electrode and served as a substrate for the FucT-catalyzed reaction. The LacNAc epitopes were converted to Lewisx (Lex) and detected by AAL. AAL binding to the Lex epitope was further confirmed in a lectin displacement and a competitive lectin binding inhibition experiment. We monitored the individual kinetic processes via EIS. The time constant for covalent immobilization of the NGP was 653 s. The FucT kinetics was the slowest process with a time constant of 1121 s. In contrast, a short time constant of 11.8 s was determined for the interaction of AAL with the modified NGPs. When this process was competed by 400 mM fucose, the binding was significantly slowed down, as indicated by a time constant of 978 s. The kinetics for the displacement of bound AAL by free fucose was observed with a time constant of 424 s. We conclude that this novel EIS biosensor and the applied workflow has the potential to detect FucT and other GT activities in general and further monitor protein-glycan interactions, which may be useful for the detection of pathogenic bacteria and cancer cells in future biomedical applications.

Different Frequency of Cyclic Tensile Strain Relates to Anabolic/Catabolic Conditions Consistent with Immunohistochemical Staining Intensity in Tenocytes.

Tenocytes are mechanosensitive cells intimately adapting their expression profile and hence, their phenotype to their respective mechanomilieu. The immunolocalization and expression intensity of tenogenic, anabolic and catabolic markers in tenocytes in response to in vitro mechanical loading have not been monitored by immunohistochemical staining (IHC). Thus, we investigated the association between IHC intensities, different stimulation frequencies, and tenogenic metabolism using a versatile mechanical stretcher. Primary tenocytes obtained from murine Achilles tendons were transferred to poly(dimethylsiloxane) (PDMS) elastomeric chamber. Chambers were cyclically stretched by 5% in uniaxial direction at a variation of tensile frequency (1 or 2 Hz) for 3 h. After stretching, cell physiology, IHC intensities of tendon-related markers, and protein level of the angiogenesis marker vascular endothelial growth factor (VEGF) were evaluated. Cell proliferation in tenocytes stimulated with 1 Hz stretch was significantly higher than with 2 Hz or without stretch, while 2 Hz stretch induced significantly reduced cell viability and proliferation with microscopically detectable apoptotic cell changes. The amount of scleraxis translocated into the nuclei and tenomodulin immunoreactivity of tenocytes treated with stretch were significantly higher than of non-stretched cells. The collagen type-1 expression level in tenocytes stretched at 1 Hz was significantly higher than in those cultivated with 2 Hz or without stretching, whereas the matrix metalloproteinase (MMP)-1 and MMP-13 immunoreactivities of cells stretched at 2 Hz were significantly higher than in those stimulated with 1 Hz or without stretching. The secreted VEGF-protein level of tenocytes stretched at 2 Hz was significantly higher than without stretching. Our IHC findings consistent with cell physiology suggest that appropriate stretching can reproduce in vitro short-term tenogenic anabolic/catabolic conditions and allow us to identify an anabolic stretching profile.

In Situ Monitoring of Membrane Protein Insertion into Block Copolymer Vesicle Membranes and Their Spreading via Potential-Assisted Approach.

Synthosomes are polymer vesicles with transmembrane proteins incorporated into block copolymer membranes. They have been used for selective transport in or out of the vesicles as well as catalysis inside the compartments. However, both the insertion process of the membrane protein, forming nanopores, and the spreading of the vesicles on planar substrates to form solid-supported biomimetic membranes have been rarely studied yet. Herein, we address these two points and, first, shed light on the real-time monitoring of protein insertion via isothermal titration calorimetry. Second, the spreading process on different solid supports, namely, SiO2, glass, and gold, via different techniques like spin- and dip-coating as well as a completely new approach of potential-assisted spreading on gold surfaces was studied. While inhomogeneous layers occur via traditional methods, our proposed potential-assisted strategy to induce adsorption of positively charged vesicles by applying negative potential on the electrode leads to remarkable vesicle spreading and their further fusion to form more homogeneous planar copolymer films on gold. The polymer vesicles in our study are formed from amphiphilic copolymers poly(2-methyl oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl oxazoline) (PMOXA-b-PDMS-b-PMOXA). Engineered variants of the transmembrane protein ferric hydroxamate uptake protein component A (FhuA), one of the largest ß-barrel channel proteins, are used as model nanopores. The incorporation of FhuA Δ1-160 is shown to facilitate the vesicle spreading process further. Moreover, high accessibility of cysteine inside the channel was proven by linkage of a fluorescent dye inside the engineered variant FhuA ΔCVFtev and hence preserved functionality of the channels after spreading. The porosity and functionality of the spread synthosomes on the gold plates have been examined by studying the passive ion transport response in the presence of Li+ and ClO4- ions and electrochemical impedance spectroscopy analysis. Our approach to form solid-supported biomimetic membranes via the potential-assisted strategy could be important for the development of new (bio-) sensors and membranes.

Effects of Plectin Depletion on Keratin Network Dynamics and Organization.

The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin ß4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and ß1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells.

Surface topography enhances differentiation of mesenchymal stem cells towards osteogenic and adipogenic lineages.

Surface topography impacts on cell growth and differentiation, but it is not trivial to generate defined surface structures and to assess the relevance of specific topographic parameters. In this study, we have systematically compared in vitro differentiation of mesenchymal stem cells (MSCs) on a variety of groove/ridge structures. Micro- and nano-patterns were generated in polyimide using reactive ion etching or multi beam laser interference, respectively. These structures affected cell spreading and orientation of human MSCs, which was also reflected in focal adhesions morphology and size. Time-lapse demonstrated directed migration parallel to the nano-patterns. Overall, surface patterns clearly enhanced differentiation of MSCs towards specific lineages: 15 µm ridges increased adipogenic differentiation whereas 2 µm ridges enhanced osteogenic differentiation. Notably, nano-patterns with a periodicity of 650 nm increased differentiation towards both osteogenic and adipogenic lineages. However, in absence of differentiation media surface structures did neither induce differentiation, nor lineage-specific gene expression changes. Furthermore, nanostructures did not affect the YAP/TAZ complex, which is activated by substrate stiffness. Our results provide further insight into how structuring of tailored biomaterials and implant interfaces - e.g. by multi beam laser interference in sub-micrometer scale - do not induce differentiation of MSCs per se, but support their directed differentiation.

Stacked planar micro coils for single-sided NMR applications.

This paper describes planar micro structured coils fabricated in a novel multilayer assembly for single-sided NMR experiments. By arranging the coil's turns in both lateral and vertical directions, all relevant coil parameters can be tailored to a specific application. To this end, we implemented an optimization algorithm based on simulations applying finite element methods (FEMs), which maximizes the coil's sensitivity and thus signal-to-noise ratio (SNR) while incorporating boundary conditions such as the coil's electrical properties and a localized sensitivity needed for single-sided applications. Utilizing thin-film technology and microstructuring techniques, the planar character is kept by a sub-millimeter overall thickness. The coils are adapted to the Profile NMR-MOUSE® magnet with a homogeneous slice of about 200 µm in height and a uniform depth gradient of about 20T/m. The final design of a coil with 20 turns, separated in four layers with five turns each, and an outer dimension of 4×4 mm(2) is able to measure a sample volume almost five times smaller than that of a state-of-the-art 14×16 mm(2) Profile NMR-MOUSE® coil with the same SNR. This allows for volume-limited measurements with high SNR and enables different future developments. The minimal dead time of 4 µs facilitates further improvements of the SNR by echo adding techniques and the characterization of samples with short T2 relaxation times. Measurements on solid polymers like polyethylene (PE) and polypropylene (PP) with T2 components as short as 200 µs approve the overall beneficial coil properties. Furthermore the ability to perform depth profiling with microscopic resolution is demonstrated.

Telemetric catheter-based pressure sensor for hemodynamic monitoring: experimental experience.

The purpose of this study was to evaluate the technical and animal experimental feasibility of a percutaneously implantable pulmonary arterial implant for permanent hemodynamic monitoring. Two systems for measuring pulmonary artery pressure (PAP) as well as pulmonary artery occlusion pressure (PAOP) were developed by modifying a commercially available pulmonary artery catheter (PAC). First, a cable-bound catheter-based system was designed by implementation of a capacitive absolute-pressure sensor in the catheter tip. This system was developed further into a completely implantable telemetric system. The devices were tested in an acute setting in a total of 10 sheep. The implant was placed with its tip in the descending pulmonary artery via the right jugular approach. Results were compared with conventional PAC positioned in the contralateral pulmonary artery using Pearson's correlation coefficients and Bland-Altman plots. Implantation of the monitoring systems was uneventful in 10 animals. Data from two fully functional cable-bound and telemetric pressure monitoring systems were available, with a total of 18,506 measurements. There was an excellent correlation between reference data and the data obtained with the implants (r = 0.9944). Bland-Altman plots indicated a very good agreement between the techniques. We report the development and successful initial test of an implantable catheter-based device for long-term measurement of PAP and PAOP. Both devices may be applicable for hemodynamic monitoring. Further long-term studies for assessing reliability and durability of the device are warranted.