Karina Mildner

Induced Pluripotent Stem Cells at Nanoscale

Reprogramming of mouse and human somatic cells into induced pluripotent stem (iPS) cells has been made possible with the expression of the transcription factor quartet Oct4, Sox2, c-Myc, and Klf4. Here, we compared iPS cells derived from mouse embryonic fibroblasts with the 4 factors to embryonic stem cells by electron microscopy. Both cell types are almost indistinguishable at the ultrastructural level, providing further evidence for the similarity of these 2 pluripotent stem cell populations.

Quantum Dots Modulate Leukocyte Adhesion and Transmigration Depending on Their Surface Modification

Although different nanosized materials, including quantum dots (QDs), are intended to be used for biomedical applications, their interactions with microvessels and their inflammatory potential are largely unknown. In this in vivo study we report that leukocyte recruitment is modulated in the presence of quantum dots. We found that the surface chemistry of QDs strongly affects their localization in postcapillary venules, their uptake by perivascular macrophages, and their potential to modify steps of leukocyte recruitment.

Surface chemistry of quantum dots determines their behavior in postischemic tissue.

The behavior of quantum dots (QDs) in the microvasculature and their impact on inflammatory reactions under pathophysiological conditions are still largely unknown. Therefore, we designed this study to investigate the fate and effects of surface-modified QDs in postischemic skeletal and heart muscle. Under these pathophysiological conditions, amine-modified QDs, but not carboxyl-QDs, were strongly associated with the vessel wall of postcapillary venules and amplified ischemia-reperfusion-elicited leukocyte transmigration. Importantly, strong association of amine-QDs with microvessel walls was also present in the postischemic myocardium. As shown by electron microscopy and verified by FACS analyses, amine-modified QDs, but not carboxyl-QDs, were associated with endogenous microparticles. At microvessel walls, these aggregates were attached to endothelial cells. Taken together, we found that both the surface chemistry of QDs and the underlying tissue conditions (i.e., ischemia-reperfusion) strongly determine their uptake by endothelial cells in microvessels, their association to endogenous microparticles, as well as their potential to modify inflammatory processes. Thus, this study strongly corroborates the view that the surface chemistry of nanomaterials and the physiological state of the tissue are crucial for the behavior of nanomaterials in vivo.

Intercellular Transport of Nanomaterials is Mediated by Membrane Nanotubes In Vivo

So-called membrane nanotubes are cellular protrusions between cells whose functions include cell communication, environmental sampling, and protein transfer. It has been previously reported that systemically administered carboxyl-modified quantum dots (cQDs) are rapidly taken up by perivascular macrophages in skeletal muscle of healthy mice. Expanding these studies, it is found, by means of in vivo fluorescence microscopy on the mouse cremaster muscle, rapid uptake of cQDs not only by perivascular macrophages but also by tissue-resident cells, which are localized more than 100 μm distant from the closest vessel. Confocal microscopy on muscle tissue, immunostained for the membrane dye DiI, reveals the presence of continuous membranous structures between MHC-II-positive, F4/80-positive cells. These structures contain microtubules, components of the cytoskeleton, which clearly colocalize with cQDs. The cQDs are exclusively found inside endosomal vesicles. Most importantly, by using in vivo fluorescence microscopy, this study detected fast (0.8 μm s(-1) , mean velocity), bidirectional movement of cQDs in such structures, indicating transport of cQD-containing vesicles along microtubule tracks by the action of molecular motors. The findings are the first to demonstrate membrane nanotube function in vivo and they suggest a previously unknown route for the distribution of nanomaterials in tissue.

The Endothelial Glycocalyx Controls Interactions of Quantum Dots with the Endothelium and Their Translocation across the Blood–Tissue Border

Advances in the engineering of nanoparticles (NPs), which represent particles of less than 100 nm in one external dimension, led to an increasing utilization of nanomaterials for biomedical purposes. A prerequisite for their use in diagnostic and therapeutic applications, however, is the targeted delivery to the site of injury. Interactions between blood-borne NPs and the vascular endothelium represent a critical step for nanoparticle delivery into diseased tissue. Here, we show that the endothelial glycocalyx, which constitutes a glycoprotein-polysaccharide meshwork coating the luminal surface of vessels, effectively controls interactions of carboxyl-functionalized quantum dots with the microvascular endothelium. Glycosaminoglycans of the endothelial glycocalyx were found to physically cover endothelial adhesion and signaling molecules, thereby preventing endothelial attachment, uptake, and translocation of these nanoparticles through different layers of the vessel wall. Conversely, degradation of the endothelial glycocalyx promoted interactions of these nanoparticles with microvascular endothelial cells under the pathologic condition of ischemia-reperfusion, thus identifying the injured endothelial glycocalyx as an essential element of the blood-tissue border facilitating the targeted delivery of nanomaterials to diseased tissue.

Integrins are required for tissue organization and restriction of neurogenesis in regenerating planarians

Tissue regeneration depends on proliferative cells and on cues that regulate cell division, differentiation, patterning and the restriction of these processes once regeneration is complete. In planarians, flatworms with high regenerative potential, muscle cells express some of these instructive cues. Here, we show that members of the integrin family of adhesion molecules are required for the integrity of regenerating tissues, including the musculature. Remarkably, in regenerating β1-integrin RNAi planarians, we detected increased numbers of mitotic cells and progenitor cell types, as well as a reduced ability of stem cells and lineage-restricted progenitor cells to accumulate at wound sites. These animals also formed ectopic spheroid structures of neural identity in regenerating heads. Interestingly, those polarized assemblies comprised a variety of neural cells and underwent continuous growth. Our study indicates that integrin-mediated cell adhesion is required for the regenerative formation of organized tissues and for restricting neurogenesis during planarian regeneration. Highlighted article: Integrin signaling acts to recruit and localize progenitor cells following injury, thereby promoting the correct organization of regenerating planarian tissue.

Influence of Surface Modifications on the Spatiotemporal Microdistribution of Quantum Dots In Vivo.

For biomedical applications of nanoconstructs, it is a general prerequisite to efficiently reach the desired target site. In this regard, it is crucial to determine the spatiotemporal distribution of nanomaterials at the microscopic tissue level. Therefore, the effect of different surface modifications on the distribution of microinjected quantum dots (QDs) in mouse skeletal muscle tissue has been investigated. In vivo real-time fluorescence microscopy and particle tracking reveal that carboxyl QDs preferentially attach to components of the extracellular matrix (ECM), whereas QDs coated with polyethylene glycol (PEG) show little interaction with tissue constituents. Transmission electron microscopy elucidates that carboxyl QDs adhere to collagen fibers as well as basement membranes, a type of ECM located on the basolateral side of blood vessel walls. Moreover, carboxyl QDs have been found in endothelial junctions as well as in caveolae of endothelial cells, enabling them to translocate into the vessel lumen. The in vivo QD distribution is confirmed by in vitro experiments. The data suggest that ECM components act as a selective barrier depending on QD surface modification. For future biomedical applications, such as targeting of blood vessel walls, the findings of this study offer design criteria for nanoconstructs that meet the requirements of the respective application.

Bleb Expansion in Migrating Cells Depends on Supply of Membrane from Cell Surface Invaginations

Cell migration is essential for morphogenesis, organ formation, and homeostasis, with relevance for clinical conditions. The migration of primordial germ cells (PGCs) is a useful model for studying this process in the context of the developing embryo. Zebrafish PGC migration depends on the formation of cellular protrusions in form of blebs, a type of protrusion found in various cell types. Here we report on the mechanisms allowing the inflation of the membrane during bleb formation. We show that the rapid expansion of the protrusion depends on membrane invaginations that are localized preferentially at the cell front. The formation of these invaginations requires the function of Cdc42, and their unfolding allows bleb inflation and dynamic cell-shape changes performed by migrating cells. Inhibiting the formation and release of the invaginations strongly interfered with bleb formation, cell motility, and the ability of the cells to reach their target.

Correlative Light and Electron Microscopy of Rare Cell Populations in Zebrafish Embryos Using Laser Marks

Correlative light and electron microscopy techniques are powerful tools that allow the identification and analysis of fluorescently labeled cells and structures of interest by an electron microscope (EM).1–3 Nevertheless, employing the methods listed above for the identification of rare cell populations can be very time consuming.4 A recently described method combines imaging of a fluorescently labeled tissue before fixation, with laser etching at the region of interest after embryo fixation.5 While the region of interest in the zebrafish embryo could be better focused on, this technique does not assist the identification of rare cell populations or specific cellular compartments when adjacent morphological landmarks are not present. In this study, we present a procedure based on a recently described method6 that allows effective identification and EM analysis of a small group of cells within the embryo, without relying on distinct morphological markers (Fig. 1). We demonstrate the merit of this procedure using zebrafish primordial germ cells, a population of less than 25 cells within early embryos, which at that developmental stage (8–12 hours post fertilization [hpf]) consist of more than 20,000 cells.7,8 This method relies on preservation of the fluorescent protein signal after fixation, combined with two-photon laser marking within the tissue at defined locations relative to the position where the cells of interest reside. FIG. 1. Schematic workflow of the correlative light and electron microscopy using laser marking. (A) Zebrafish embryo fixed in 2% PFA + 0.2% glutaraldehyde in 0.1 M PHEM buffer for 10 min, deyolked and fixed for an additional 2 h ...

Cryo-Immuno Electron Microscopy of Peroxisomal Marker Proteins

Electron microscopy samples processed for cryo-immunogold-labeling need to be gently fixed to keep their antigenicity. Biological material like cultured cells or tissue can be prepared according to the standard Tokuyasu fixation or in a further developed rehydration method based on high-pressure freezing. We will describe here the variant and common steps of both methods in detail and illustrate their potency in the ultrastructural imaging of peroxisomes.