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. As a proof of concept, a microfluidic fusion chip embodied with a microslit (4 µm in width) to prevent nuclei mixing was developed and used to perform one-to-one electrofusion of a target somatic cell (Jurkat cell) with an induced pluripotent stem (iPS) cell. To extract its cytoplasm, the target cell was first topologically connected to a sacrificial iPS cell by electrofusion via a microslit, followed by shear flow removal of the latter to obtain a cytoplasm-depleted nucleus of the target cell. Then, to replace the lost cytoplasm, topological reconnection to a second iPS cell was performed similarly by electrofusion, followed by shear flow separation of the target cell to enable it acquire most of the iPS cytoplasm, but without nuclei mixing. Microscopic observation of target cells harvested and cultured post hoc in a microwell confirmed that they manifested cell division. Taken together, these results demonstrate the potential application of the cell-cell topological reconnection technique to somatic cell nuclear transplantation for the generation of autologous pluripotent stem cells.

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. In the present study, we developed a microfluidic device that enables non-destructive isolation of chromosomes from single mammalian cells as well as real-time microscopic monitoring of the partial unfolding and stretching of individual chromosomes with increasing salt concentrations under a gentle flow. Using this device, we compared the folding stability of chromosomes between non-differentiated and differentiated cells and found that the salt concentration which induces the chromosome unfolding was lower (≤500 mM NaCl) for chromosomes derived from undifferentiated cells, suggesting that the chromatin folding stability of these cells is lower than that of differentiated cells. In addition, individual unfolded chromosomes, i.e., chromatin fibres, were stretched to 150-800 µm non-destructively under 750 mM NaCl and showed distributions of highly/less folded regions along the fibres. Thus, our technique can provide insights into the aspects of chromatin folding that influence the epigenetic control of cell differentiation.

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. To modulate the adhesion microenvironment, we used the mesh culture technique recently developed by our group, which involves culturing hiPSCs on suspended micro-structured meshes with limited surface area for cell adhesion. We show that this adhesion-restriction strategy can trigger a two-stage self-organization of hiPSCs; first into stem cell sheets, which express pluripotency signatures until around day 8-10, then into spherical cysts following differentiation and self-organization of the sheet-forming cells. Detailed morphological analysis using immunofluorescence microscopy with both confocal and two-photon microscopes revealed the anatomy of the cysts as consisting of a squamous epithelial wall richly expressing E-cadherin and CDX2. We also confirmed that the cysts exhibit a polarized morphology with basal protrusions, which show migratory behavior when anchored. Together, our results point to the formation of cysts which morphologically resemble the trophectoderm at the late-stage blastocyst. Thus, the mesh culture microenvironment can initiate self-organization of hiPSCs into trophectoderm-mimicking cysts as organoids with potential application in the study of early embryogenesis and also in drug development.

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. An appropriate combination of canonical and non-canonical histones, also known as histone variants, allows each cell to exert its cell type-specific functions. Here, we aimed to reveal the dynamics of histone usage and chromatin structure during the differentiation of trophoblast stem cells (TSCs) into TGCs. Although the expression of most genes encoding canonical histones was downregulated, the expression of a few genes encoding histone variants such as H2AX, H2AZ, and H3.3 was maintained at a relatively high level in TGCs. Both the micrococcal nuclease digestion assay and nucleosome stability assay using a microfluidic device indicated that chromatin became increasingly loose as TSCs differentiated. Combinatorial experiments involving H3.3-knockdown and -overexpression demonstrated that variant H3.3 resulted in the formation of loose nucleosomes in TGCs. In conclusion, our study revealed that TGCs possessed loose nucleosomes owing to alterations in their histone composition during differentiation.

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. To this end, the authors developed a microfluidic platform that enabled us to isolate chromatin fibers from single cells and tether them to microstructures in a microfluidic channel without fragmentation. The fibers were then exposed to varying concentrations of salt solution under microscopic observation. As a result, the fibers are non-uniformly elongated by up to 2-3 times along the fiber axis as salt concentration was increased from 0 to 3 M, suggesting that chromosome structural stability is non-uniformly distributed along chromatin fibers in their native form. Thus, our system enables direct microscopic analysis of individual chromatin fibers from single cells, which can provide insights into epigenetic mechanisms of cell development, cell differentiation, and carcinogenesis.

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. The success rate and precision of the fabrications were evaluated. The results indicate that the microstructures are obtained in an aqueous solution with a precision ∼50 nm at concentrations in the order of 10(6) particles/ml. The visibility of these microstructures under a fluorescence microscope was also characterised, along with the elucidation of the fabrication parameters needed to fine tune visibility. Manipulating yeast chromosomal DNA molecules with the microstructures illustrated the relationship between the efficiency of manipulation and the geometrical shape of the microstructure. This report provides the guidelines for designing microstructures used in single DNA molecule analysis based on on-site DNA manipulation, and is expected to broaden the applications of this technique in the future.

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. We used microfabricated mesh sheets that consisted of open meshes (100∼200 µm in pitch) with narrow mesh strands (3-5 µm in width) to provide support for initial cell attachment and growth. We demonstrate that minimization of cell adhesion area achieved by this culture method can trigger a sequence of morphogenetic transformations that begin with individual hiPSCs attached on the mesh strands proliferating to form cell sheets by self-assembly organization and ultimately differentiating after 10-15 days of mesh culture to generate spherical cysts that secreted human chorionic gonadotropin (hCG) hormone and expressed caudal-related homeobox 2 factor (CDX2), a specific marker of trophoblast lineage. Thus, this study demonstrates a simple and direct mechanical approach to induce trophoblast differentiation and generate cysts for application in the study of early human embryogenesis and drug development and screening.

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. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca(2+)] change in the ß-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of ß-cells.

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. For the non-destructive handling of the isolated chromatin fibers in the microfluidic device, we developed antibody-conjugated microspheres, which had affinity to the fluorescent protein-tagged core histones, and the microspheres were manipulated using optical tweezers, which functioned as optically driven microtools. With the aid of the microtool, isolated chromatin fibers were handled non-destructively and were tethered at the microstructures fabricated in the microfluidic device with straightened conformation by the flow. Immunofluorescence staining was carried out as a demonstrative biochemical experiment with the individual native chromatin fibers isolated in the microfluidic device, and specific fluorescent spots were visualized along the tethered chromatin fibers. Thus, the potential application of this method for epigenetic analyses of intact chromatin fibers isolated from single cells is demonstrated.

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. This paper reviews some of our research, including gene transfection, electrofusion and cytoplasmic transplant.

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. λ DNA, which was used as the target DNA, was stretch-and-positioned onto microelectrodes by using the electrostatic method. When the ssDNA-RecA complex was fed to the suspended target DNA, clear fluorescence spots were observed on individual target DNA molecules. The histogram of the probe-binding position along the target DNA was measured, and the peak was found to correspond to the location complementary to the probe ssDNA. This result shows a potential use for recombination proteins in facilitating the optical detection of DNA sequences.

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. The denaturation was detected in real time by separating quantum dots on each strand. Results showed that complete separation of the strands occurred within a few seconds of laser irradiation, which raised the temperature to approximately 90 °C.

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. When a pulse voltage is applied to the electrodes, most voltage drop occurs around the orifice and impressed on the cell membrane in the orifice. This makes possible the application of size-independent voltage to fuse two cells in contact at all orifices exclusively in 1:1 manner. In the experiment, cytoplasm of one of the cells is stained with a fluorescence dye, and the transfer of the fluorescence to the other cell is used as the indication of fusion events. The two-dimensional orifice arrangement at the pitch of 50 µm realizes simultaneous fusion of 6 × 10³ cells on a 4 mm diameter chip, and the fusion yield of 78-90% is achieved for various sizes and types of cells.

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. We fabricated a microfluidic chip that contained a chamber and partition. The partition divided the chamber into two compartments and it had a number of embedded micro-orifices. The voltage applied to the electrodes located at each compartment generated an electric field distribution concentrating in micro-orifices. Cells introduced into each compartment moved toward the micro-orifice array by manipulation of hydrostatic pressure. DEP assisted trapping was used to keep the cells in micro-orifice and to establish cell to cell contact through orifice. By applying a pulse, cell fusion was initiated to form a neck between cells. The neck passing through the orifice resulted in immobilization of the fused cell pair at micro-orifice. After washing away the unfused cells, the chip was loaded to a microscope with stage top incubator for time lapse imaging of the selected fusants. The viable fusants were successfully generated by fusion of mouse fibroblast cells (L929). Time lapse observation of the fusants showed that fused cell pairs escaping from micro-orifice became one tetraploid cell. The generated tetraploid cells divided into three daughter cells. The fusants generated with a smaller micro-orifice (diameter approximately 2 mum) were kept immobilized at micro-orifice until cell division phase. After observation of two synchronized cell divisions, the fusant divided into four daughter cells. We conclude that the presented method of cell pairing and fusion is suitable for high-yield generation of viable fusants and furthermore, subsequent study of postfusion phenomena.

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.

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.