Thasaneeya Kuboki

Time-dependent migratory behaviors in the long-term studies of fibroblast durotaxis on a hydrogel substrate fabricated with a soft band.

Durotaxis, biased cell movement up a stiffness gradient on culture substrates, is one of the useful taxis behaviors for manipulating cell migration on engineered biomaterial surfaces. In this study, long-term durotaxis was investigated on gelatinous substrates containing a soft band of 20, 50, and 150 μm in width fabricated using photolithographic elasticity patterning; sharp elasticity boundaries with a gradient strength of 300 kPa/50 μm were achieved. Time-dependent migratory behaviors of 3T3 fibroblast cells were observed during a time period of 3 days. During the first day, most of the cells were strongly repelled by the soft band independent of bandwidth, exhibiting the typical durotaxis behavior. However, the repellency by the soft band diminished, and more cells crossed the soft band or exhibited other mixed migratory behaviors during the course of the observation. It was found that durotaxis strength is weakened on the substrate with the narrowest soft band and that adherent affinity-induced entrapment becomes apparent on the widest soft band with time. Factors, such as changes in surface topography, elasticity, and/or chemistry, likely contributing to the apparent diminishing durotaxis during the extended culture were examined. Immunofluorescence analysis indicated preferential collagen deposition onto the soft band, which is derived from secretion by fibroblast cells, resulting in the increasing contribution of haptotaxis toward the soft band over time. The deposited collagen did not affect surface topography or surface elasticity but did change surface chemistry, especially on the soft band. The observed time-dependent durotaxis behaviors are the result of the mixed mechanical and chemical cues. In the studies and applications of cell migratory behavior under a controlled stimulus, it is important to thoroughly examine other (hidden) compounding stimuli in order to be able to accurately interpret data and to design suitable biomaterials to manipulate cell migration.

Target of rapamycin (TOR) controls vitellogenesis via activation of the S6 kinase in the fat body of the tick, Haemaphysalis longicornis.

Vitellogenin (Vg) synthesis, vitellogenesis, is an essential process for the development and reproduction of ticks. Our previous finding led to the hypothesis that target of rapamycin (TOR) pathway is important for vitellogenesis in the hard tick, Haemaphysalis longicornis. The TOR pathway controls cellular activity according to nutrient availability in eukaryotes. TOR, a member of the phosphatidylinositol 3-kinase family, is a central player in this pathway. Here, we present preliminary evidence that H. longicornis TOR (HlTOR) controls vitellogenesis via activation of S6 kinase (S6K) in the fat body. RNA interference (RNAi)-mediated gene silencing of HlTOR was undertaken to elucidate the involvement of HlTOR in the vitellogenesis of the tick. HlTOR-RNAi caused inhibition of S6K phosphorylation in the fat body. HlTOR-RNAi also altered not only the expression levels of GATA mRNA and protein but also the intracellular localisation of GATA in the fat body. The expression levels of Vg mRNA and protein in the fat body of HlTOR-RNAi ticks were significantly lower than those in control ticks. In the pre-ovipositional stage, the ovaries of control ticks had brown oocytes developing, but those of HlTOR-RNAi ticks were white and immature. The haemolymph colour indicated that the amount of Vg was lower in HlTOR-RNAi ticks than in the controls. Furthermore, rapamycin inhibited S6K phosphorylation and reduced the expression levels of Vg mRNA and protein in the fat bodies. Vg proteins were not detected in rapamycin-treated fat bodies in the presence of 20-hydroxyecdysone. These results suggest that HlTOR activity is critical for vitellogenesis stimulated by 20-hydroxyecdysone.

High-resolution imaging of a cell-attached nanointerface using a gold-nanoparticle two-dimensional sheet

This paper proposes a simple, effective, non-scanning method for the visualization of a cell-attached nanointerface. The method uses localized surface plasmon resonance (LSPR) excited homogeneously on a two-dimensional (2D) self-assembled gold-nanoparticle sheet. The LSPR of the gold-nanoparticle sheet provides high-contrast interfacial images due to the confined light within a region a few tens of nanometers from the particles and the enhancement of fluorescence. Test experiments on rat basophilic leukemia (RBL-2H3) cells with fluorescence-labeled actin filaments revealed high axial and lateral resolution even under a regular epifluorescence microscope, which produced higher quality images than those captured under a total internal reflection fluorescence (TIRF) microscope. This non-scanning-type, high-resolution imaging method will be an effective tool for monitoring interfacial phenomena that exhibit relatively rapid reaction kinetics in various cellular and molecular dynamics.

Persistent random deformation model of cells crawling on a gel surface

In general, cells move on a substrate through extension and contraction of the cell body. Though cell movement should be explained by taking into account the effect of such shape fluctuations, past approaches to formulate cell-crawling have not sufficiently quantified the relationship between cell movement (velocity and trajectory) and shape fluctuations based on experimental data regarding actual shaping dynamics. To clarify this relationship, we experimentally characterized cell-crawling in terms of shape fluctuations, especially extension and contraction, by using an elasticity-tunable gel substrate to modulate cell shape. As a result, an amoeboid swimmer-like relation was found to arise between the cell velocity and cell-shape dynamics. To formulate this experimentally-obtained relationship between cell movement and shaping dynamics, we established a persistent random deformation (PRD) model based on equations of a deformable self-propelled particle adopting an amoeboid swimmer-like velocity-shape relationship. The PRD model successfully explains the statistical properties of velocity, trajectory and shaping dynamics of the cells including back-and-forth motion, because the velocity equation exhibits time-reverse symmetry, which is essentially different from previous models. We discuss the possible application of this model to classify the phenotype of cell migration based on the characteristic relation between movement and shaping dynamics.

LSPR-mediated high axial-resolution fluorescence imaging on a silver nanoparticle sheet

This paper reports our original technique for visualizing cell-attached nanointerfaces with extremely high axial resolution using homogeneously excited localized surface plasmon resonance (LSPR) on self-assembled silver nanoparticle sheets. The LSPR sheet can confine and enhance the fluorescence at the nanointerface, which provides high signal-to-noise ratio images of focal adhesion at the cell-attached interface. The advantage of this LSPR-assisted technique is its usability, which provides comparable or higher-quality nanointerfacial images than TIRF microscopy, even under epifluorescence microscopy. We also report the cytotoxicity of silver nanoparticles, as determined via morphological analysis of adherent cells on the sheet.

Reprogramming of mouse fibroblasts into neural lineage cells using biomaterials.

Introduction: Induced neural stem cells (iNSCs) have the ability of differentiation into neurons, astrocytes and oligodendrocytes. iNSCs are very useful in terms of research and treatment. The present study offers an idea that biomaterials could be one of the tools that could modulate reprogramming process in the fibroblasts. Methods: Gelatin biomaterials were fabricated into 3 types, including (i) gelatin, (ii) gelatin with 1 mg/mL hydroxyapatite, and (iii) gelatin with hydroxyapatite and pig brain. NIH/3T3 fibroblasts were cultured on each type of biomaterial for 7, 9 and 14 days. RT-PCR was performed to investigate the gene expression of the fibroblasts on biomaterials compared to the fibroblasts on tissue culture plates. PI3K/Akt signaling was performed by flow cytometry after 24 hours seeding on the biomaterials. The biomaterials were also tested with the human APCs and PDL cells. Results: The fibroblasts exhibited changes in the expression of the reprogramming factor; Klf‫4 and the neural transcription factors; NFIa, NFIb and Ptbp1 after 9 days culture. The cultivation of fibroblasts on the biomaterials for 7 days showed a higher expression of the transcription factor SOX9. The expression of epigenetic genes; Kat2a and HDAC3 were changed upon the cultivation on the biomaterials for 9 days. The fibroblasts cultured on the biomaterials showed an activation of PI3K/Akt signaling. The human APCs and human PDL cells developed mineralization process on biomaterials Conclusion: Changes in the expression of Klf4, NFIa, NFIb, Ptbp1 and SOX9 indicated that fibroblasts were differentiated into an astrocytic lineage. It is possible that the well-designed biomaterials could work as powerful tools in the reprogramming process of fibroblasts into iNSCs.

Fabrication of Elasticity-Tunable Gelatinous Gel for Mesenchymal Stem Cell Culture.

Surface elasticity or stiffness of an underlying substrate may regulate cellular functions such as adhesion, proliferation, signaling, differentiation, and migration. Recent studies have reported on the development of biomaterials to control stem cell fate determination via the stiffness of the culture substrates. In this chapter, we provide a detailed protocol for fabricating elasticity-tunable gelatinous hydrogels for stem cell culture with photo-induced or thermo-induced crosslinking of well-developed styrenated gelatin (StG). We also include the detailed application of gelatinous gel for mesenchymal stem cell (MSC) culture and sample collection for transcriptional and proteomic analysis.