Nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system.

Previous studies have demonstrated that somatic cells fused with pluripotent stem cells can be reprogrammed on the basis of reprogramming factors acquired from the latter. However, fusion-reprogrammed cells are deemed unsuitable for therapeutic applications mainly because conventional fusion techniques often yield tetraploid fusants that contain exogenous genes acquired from the fusion partners. Here, we present a novel cell-cell topological reconnection technique and demonstrate its application to nuclear transplantation between a somatic cell and a stem cell without nuclei mixing.

A microfluidic device for isolating intact chromosomes from single mammalian cells and probing their folding stability by controlling solution conditions.

Chromatin folding shows spatio-temporal fluctuations in living undifferentiated cells, but fixed spatial heterogeneity in differentiated cells. However, little is known about variation in folding stability along the chromatin fibres during differentiation. In addition, effective methods to investigate folding stability at the single cell level are lacking.

Nucleosomes of polyploid trophoblast giant cells mostly consist of histone variants and form a loose chromatin structure.

Trophoblast giant cells (TGCs) are one of the cell types that form the placenta and play multiple essential roles in maintaining pregnancy in rodents. TGCs have large, polyploid nuclei resulting from endoreduplication. While previous studies have shown distinct gene expression profiles of TGCs, their chromatin structure remains largely unknown.

Self-organization of human iPS cells into trophectoderm mimicking cysts induced by adhesion restriction using microstructured mesh scaffolds.

Cellular dynamics leading to the formation of the trophectoderm in humans remain poorly understood owing to limited accessibility to human embryos for research into early human embryogenesis. Compared to animal models, organoids formed by self-organization of stem cells in vitro may provide better insights into differentiation and complex morphogenetic processes occurring during early human embryogenesis. Here we demonstrate that modulating the cell culture microenvironment alone can trigger self-organization of human induced pluripotent stem cells (hiPSCs) to yield trophectoderm-mimicking cysts without chemical induction.

Nucleosomes Exhibit Non-uniform Unwrapping Along Native Chromatin Fibers with Increasing Salt Concentration as Revealed by Direct Imaging in a Microfluidic Channel.

Identifying the distribution of the higher-order structure of chromatin - a complex of DNA and proteins - along genomic DNA can clarify the mechanisms underlying cell development and differentiation, including gene regulation. However, genome-wide analysis of this distribution at the single-cell level remains an outstanding challenge. Here, the authors report a new method for investigating changes in and the distribution of higher-order structures along native chromatin fibers - ranging over 100 µm in length - relative to changes in salt concentration.

Adhesion patterning by a novel air-lock technique enables localization and real-time imaging of reprogramming events in one-to-one electrofused hybrids.

Although fusion of somatic cells with embryonic stem (ES) cells has been shown to induce reprogramming, single-cell level details of the transitory phenotypic changes that occur during fusion-based reprogramming are still lacking. Our group previously reported on the technique of one-to-one electrofusion via micro-slits in a microfluidic platform. In this study, we focused on developing a novel air-lock patterning technique for creating localized adhesion zones around the micro-slits for cell localization and real-time imaging of post fusion events with a single-cell resolution.

Characterisation of optically driven microstructures for manipulating single DNA molecules under a fluorescence microscope.

Optical tweezers are powerful tools for manipulating single DNA molecules using fluorescence microscopy, particularly in nanotechnology-based DNA analysis. We previously proposed a manipulation technique using microstructures driven by optical tweezers that allows the handling of single giant DNA molecules of millimetre length that cannot be manipulated by conventional techniques. To further develop this technique, the authors characterised the microstructures quantitatively from the view point of fabrication and efficiency of DNA manipulation under a fluorescence microscope.

Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells.

Mechanical methods for inducing differentiation and directing lineage specification will be instrumental in the application of pluripotent stem cells. Here, we demonstrate that minimization of cell-substrate adhesion can initiate and direct the differentiation of human pluripotent stem cells (hiPSCs) into cyst-forming trophoblast lineage cells (TLCs) without stimulation with cytokines or small molecules. To precisely control cell-substrate adhesion area, we developed a novel culture method where cells are cultured on microstructured mesh sheets suspended in a culture medium such that cells on mesh are completely out of contact with the culture dish.

Gallbladder lymphoma in a miniature dachshund.

A 7-year-old, miniature dachshund was referred for examination and treatment of persistent anorexia, deep yellow-coloured urine and leucocytosis. The clinical sign of jaundice, results from a serum biochemistry profile and ultrasonographic images suggested a biliary tract obstruction. A cholecystectomy was performed to remove the obstruction.

Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells.

In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution.

Non-destructive handling of individual chromatin fibers isolated from single cells in a microfluidic device utilizing an optically driven microtool.

We report a novel method for the non-destructive handling of, and biochemical experiments with, individual intact chromatin fibers, as well as their isolation from single cells, utilizing a specifically designed microfluidic device with an optically driven microtool under the microscope. Spheroplasts of recombinant fission yeast cells expressing fluorescent protein-tagged core histones were employed, and isolation of chromatin fibers was conducted by cell bursting via changing from isotonic conditions to hypotonic conditions in the microfluidic device. The isolation of chromatin fibers was confirmed by the fluorescent protein-tagged core histones involved in the chromatin fibers.

Electroporation and electrofusion in field-tailored microstructures.

Bringing foreign substances into cells is a basic process in cell engineering. So called reversible breakdown of the cell membrane by electrical pulses opens up transient pores on the membrane through which molecules can diffuse into, or contacting cells can fuse. Using micro-fabricated structures with the dimension smaller than that of cells, the field pattern can be designed and tailored, that enables the handling of single cells, as well as the control over the location and the magnitude of the membrane voltage to achieve low invasive high-yield poration or fusion.

Optical sequence probing with the homologous recombination protein RecA.

In this study, we used the homologous recombination protein RecA to locate a specific sequence on DNA. Single-stranded (ss) DNA (80-mer, 5'-biotinylated), complementary to the sequence of interest, was labeled with quantum dots (Qdots(®)) via biotin-avidin binding. The DNA was then mixed with RecA to form a fluorescent-labeled ssDNA-RecA complex.

Portal vein aneurysm in a dog.

Portal vein aneurysm (PVA) is a rare abnormal dilatation of the portal vein, which has not been reported in dogs. We describe the findings of ultrasound and computed tomography in a case of PVA in a young male toy poodle, with the final diagnosis established by explorative surgical observation. The dog had an aneurysmal fusiform dilatation in the extrahepatic portal vein with portal hypertension and multiple portsystemic shunts.

Single DNA molecule denaturation using laser-induced heating.

This paper proposes targeted in situ denaturation through laser-induced heating to partially amplify relevant sequences from a long DNA strand. It uses 5 kb of DNA as a sample, labeling both strands with quantum dots, with one strand immobilized on a solid surface. We irradiated a targeted DNA sequence with a focused infrared laser to elevate its temperature, monitoring the process by microscope.

Folding dynamics of tethered giant DNA under strong flow.

Using a microfluidic device, we investigate the folding dynamics of individual linear long DNA, whose one end is tethered under a strong flow in the presence of a condensing agent. Direct observations of the folding process of DNA molecules reveal a characteristic dynamics with pronounced non-monotonic velocity of the folded part at the free end against the flow. We discuss this unique dynamics in relation to the inhomogeneous spatial fluctuation and the structure change at the multiple order levels along the stretched DNA, which is induced by the increasing tension due to the build-up of the hydrodynamic drag force.

Dielectrophoresis-assisted massively parallel cell pairing and fusion based on field constriction created by a micro-orifice array sheet.

In this paper, we present a novel electrofusion device that enables massive parallelism, using an electrically insulating sheet having a two-dimensional micro-orifice array. The sheet is sandwiched by a pair of micro-chambers with immersed electrodes, and each chamber is filled with the suspensions of the two types of cells to be fused. Dielectrophoresis, assisted by sedimentation, is used to position the cells in the upper chamber down onto the orifices, then the device is flipped over to position the cells on the other side, so that cell pairs making contact in the orifice are formed.

Multiple cases of cutaneous Mycobacterium massiliense infection in a "hot spa" in Japan.

Seven body polishers working in the same "hot spa" presented with multiple red nodules and papules on their hands and forearms. A causative agent was successfully isolated from two of the subjects and from a swab sample collected from the underside of a bed cover in the body-polishing facility. The two cutaneous isolates and the environmental isolate were rapidly growing mycobacteria that formed nonphotochromogenic smooth or smooth/rough colonies on Ogawa egg slants.

Dielectrophoretic cell trapping and parallel one-to-one fusion based on field constriction created by a micro-orifice array.

Micro-orifice based cell fusion assures high-yield fusion without compromising the cell viability. This paper examines feasibility of a dielectrophoresis (DEP) assisted cell trapping method for parallel fusion with a micro-orifice array. The goal is to create viable fusants for studying postfusion cell behavior.

Microorifice-based high-yield cell fusion on microfluidic chip: electrofusion of selected pairs and fusant viability.

Microorifice-based fusion makes use of electric field constriction to assure high-yield one-to-one fusion of selected cell pairs. The aim of this paper is to verify feasibility of high-yield cell fusion on a microfluidic chip. This paper also examines viability of the fusant created on the chip.

Spontaneous formation of giant unilamellar vesicles from microdroplets of a polyion complex by thermally induced phase separation.

Water pump: Polyion complex (PIC) vesicles are spontaneously formed from PIC microdroplets, which are formed by mixing cationic and anionic polymers (see picture). The formation process can be reversibly controlled by local heating with a focused infrared laser that triggers microphase separation and subsequent water influx. The size of the resulting giant unilamellar vesicles is determined by the initial size of the PIC droplets.

Measurement of brain trace elements in a dog with a portosystemic shunt: relation between hyperintensity on T1-weighted magnetic resonance images in lentiform nuclei and brain trace elements.

Prior to euthanasia, brain magnetic resonance imaging (MRI) was performed for a five-year-old male Yorkshire Terrier following portosystemic shunt (PSS) surgical attenuation. Hyperintensity was observed on T1W images of the lentiform nuclei. Trace elements in this area were measured by inductively coupled plasma atomic emission spectrometry.

High-yield electrofusion of biological cells based on field tailoring by microfabricated structures.

The authors present the use of electric-field constriction created by a microfabricated structure to realise high-yield electrofusion of biological cells. The method uses an orifice on an electrically insulating wall (orifice plate) whose diameter is as small as that of the cells. Owing to the field constriction created by the orifice, we can induce the controlled magnitude of membrane voltage selectively around the contact point, regardless of the cell size.

Polarisation and membrane voltage of ellipsoidal particle with a constant membrane thickness: a series expansion approach.

The estimation of the membrane voltage and the polarisation factor of biological cells provide a base for the study of bio-manipulation techniques, such as dielectrophoresis, electroporation or electrofusion. To model a biological cell, an ellipsoidal particle with an insulating membrane is sometimes employed, but due to the limitation of the confocal nature of the coordinate system, the membrane thickness is assumed to vary with the position, despite the fact that the lipid bilayer membrane has a uniform thickness. The authors present a method to rigorously treat the uniform-thickness condition in a system having an axial symmetry.

On-site manipulation of single chromosomal DNA molecules by using optically driven microstructures.

We report a novel method for manipulation of single giant DNA molecules under a video microscope. Using optically driven microstructures, we manipulated chromosomal DNA of length in the order of millimetres, extended by electroosmotic flow without DNA breakage in aqueous solution: we picked up DNA, using microfabricated hooks and wound it around microfabricated bobbins.