Kazuyoshi Itoga

Hippo pathway regulation by cell morphology and stress fibers

The Hippo signaling pathway plays an important role in regulation of cell proliferation. Cell density regulates the Hippo pathway in cultured cells; however, the mechanism by which cells detect density remains unclear. In this study, we demonstrated that changes in cell morphology are a key factor. Morphological manipulation of single cells without cell-cell contact resulted in flat spread or round compact cells with nuclear or cytoplasmic Yap, respectively. Stress fibers increased in response to expanded cell areas, and F-actin regulated Yap downstream of cell morphology. Cell morphology- and F-actin-regulated phosphorylation of Yap, and the effects of F-actin were suppressed by modulation of Lats. Our results suggest that cell morphology is an important factor in the regulation of the Hippo pathway, which is mediated by stress fibers consisting of F-actin acting upstream of, or on Lats, and that cells can detect density through their resulting morphology. This cell morphology (stress-fiber)-mediated mechanism probably cooperates with a cell-cell contact (adhesion)-mediated mechanism involving the Hippo pathway to achieve density-dependent control of cell proliferation.

Fabrication of transferable micropatterned-co-cultured cell sheets with microcontact printing

The purpose of the present study is to develop a novel method for the fabrication of transferable micropatterned cell sheets for tissue engineering. To achieve this development, microcontact printing of fibronectin on commercially available temperature-responsive dishes was employed. Primary rat hepatocytes were seeded on the dish surfaces printed with fibronectin. Under serum-free conditions, hepatocytes were attached onto fibronectin domains selectively. Then, a second cell type of endothelial cells was seeded in the presence of serum. Double fluorescent staining revealed that endothelial cells successfully adhered to the intervals of hepatocyte domains. Finally, all the cells were harvested as a single contiguous micropatterned cell sheet upon temperature-reduction. With a cell sheet manipulator having a gelatin layer for the support of harvested cell sheets, harvested micropatterned cell sheets were transferred to new dish surfaces. This technique would be useful for the fabrication of thick tissue constructs having a complex microarchitecture.

Mass preparation of size-controlled mouse embryonic stem cell aggregates and induction of cardiac differentiation by cell patterning method

Embryonic stem cells (ESCs) are promising cell sources for cell-based therapy. It has been established that the formation of ESC aggregates promotes their differentiation into the derivatives of all three germ layers. ESC aggregates are generally prepared via the formation of suspended spherical aggregates called embryoid bodies (EBs). Because the differentiation efficiency depends on the size of EBs, it becomes one of the research topics how to prepare size-controlled EBs in a scalable manner for reproducible and high-throughput experiments. Here, we have developed a novel culture method that enables simple mass preparation of size-controlled ESC aggregates on a culture surface instead of floating EBs. We developed a maskless photolithography device that enabled rapid fabrication of micropatterned surfaces. Utilizing this device, we fabricated the culture substrates the surfaces of which comprised arrays of cell-adhesive circular micro-domains (100-400 microm in diameter) and the rest of non-cell-adhesive domains. We seeded mouse ESCs on this substrate and prepared size-controlled ESC aggregates on the micro-domains. We analyzed cardiac differentiation in the ESC aggregates and found that the optimal diameter of micro-domains was 200 microm. The present method is useful for the simple and reproducible mass preparation of ESC-derived differentiated cells and high-throughput assays.

Second-Generation Maskless Photolithography Device for Surface Micropatterning and Microfluidic Channel Fabrication

We have previously reported on a maskless photolithography device for surface micropatterning and microfabrication by modifying a commercially available liquid crystal display projector. For the prototype, 10-microm resolution was achieved by downsizing the image on a 0.7-in. liquid crystal display panel to an area of 8 x 6 mm and projecting it on a fixed stage. Here, we report on a second-generation maskless photolithography device having two novel features. First, the sliding lens system with variable focal distances and exchangeable objective lenses achieves a variable resolution of 2-8 mum. Second, the synchronous control of displayed images generated by a personal computer and the movement of a XY-positioning stage allows for the fabrication of micropatterns over a larger area (over 50 x 50 mm). Here, we show examples fabricated with the two novel features.

The co-application effects of fullerene and ascorbic acid on UV-B irradiated mouse skin.

The role of fullerene as a pro-oxidant or anti-oxidant in Ultraviolet B ray (UV-B)-induced disorders in mouse skin was investigated. Fullerene gave no photo-toxic effect to UV-B-irradiated mouse skin. Since erythema was concentrated at the pore circumference in a UV-B irradiation experiment in mouse skin, the sebaceous gland pairs was strongly implicated as a site for the generation of reactive oxygen species (ROS). In a histological evaluation of the skin stained with CH(3)MDFDA (ROS index) and YO-Pro-1 (apoptosis index), the fluorescence intensity of a sebaceous gland significantly increased with UV-B irradiation. With the application of fullerene to UV-irradiated mouse skin, no toxicity was recognized in comparison with the control, and erythema, the ROS index, and the apoptosis index decrease with the application of fullerene. Ascorbyl radical (AA*) increased with the application of ascorbate (AA) to UV-B-irradiated mouse skin, and AA* decreased with the application of fullerene. The co-application of AA and fullerene, which suppressed AA* in vitro, significantly suppressed erythema, and also suppressed both the ROS index and apoptosis index in mouse skin after UV-B irradiation. In both mouse skin at 48 h after UV-B irradiation and in an attempt to reproduce this phenomenon artificially in vitro, a similar high AA* peak (AA*/H*>4) was observed in electron spin resonance (ESR) charts. The binding of fullerene with AA impairs the Fenton reaction between AA and Fe-protein based on the observation of ascorbate-specific UV absorption and a linear equation for the calibration curve. Therefore, fullerene may impair the intercalation of AA to a heme pocket by binding with AA. These results suggest that the co-application of AA and fullerene is effective against oxidative skin damage caused by UV-B irradiation, and the development of an AA* inhibitor such as fullerene should be useful for reducing organ damage associated with Fe-protein oxidation.

The Mechanism of Melanocytes-Specific Cytotoxicity Induced by Phenol Compounds Having a Prooxidant Effect, relating to the Appearance of Leukoderma

Specific phenol compounds including rhododendrol (RD), a skin-brightening ingredient in cosmetics, are reported to induce leukoderma, inducing a social problem, and the elucidation of mechanism of leukoderma is strongly demanded. This study investigated the relationship among the cytotoxicities of six phenol compounds on B16F10 melanoma cells and HaCaT keratinocytes and generated reactive oxygen species (ROS). As a result, the cytotoxicity of RD on B16F10 cells was higher than that on HaCaT cells, and RD significantly increased intracellular ROS and hydrogen peroxide (H2O2) levels in B16F10 cells. Furthermore, although raspberry ketone (RK), RD derivative, also increased intracellular ROS in B16F10 cells, increase in ROS was suppressed by disodium dihydrogen ethylenediaminetetraacetate dehydrate (EDTA). The amounts of increased ROS with RK in HaCaT cells without melanocyte were further increased by tyrosinase. Therefore, tyrosinase, a metalloprotein having copper, was speculated to be one of causative agents allowing phenol compounds to work as a prooxidant. Hydroxyl radical was generated by adding a mixture of tyrosinase and H2O2 to RD, and the amount of the radical was further increased by UVB, indicating that RD cytotoxicity was caused by intracellularly increased ROS, which possibly related to phenol induced prooxidants.

Rate control of cell sheet recovery by incorporating hydrophilic pattern in thermoresponsive cell culture dish

Thready stripe-polyacrylamide (PAAm) pattern was fabricated on a thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) surface, and their surface properties were characterized. A PIPAAm surface spin-coated with positive photoresist was irradiated through a 5 µm/5 µm or a 10 µm/10-µm black and white striped photomask, resulting in the radical polymerization of AAm on the photoirradiated area. After staining with Alexa488 bovine serum albumin, the stripe-patterned surface was clearly observed and the patterned surface was also observed by a phase contrast image of an atomic force microscope. NIH-3T3 (3T3) single cells were able to be cultured at 37°C on the patterned surfaces as well as on a PIPAAm surface without pattern, and the detachment of adhered cells was more rapidly from the patterned surface after reducing temperature. Furthermore, the rate of detachment of 3T3 confluent cell sheet on the patterned surface was accelerated, compared with on a conventional PIPAAm surface under the static condition. The rate control of cell sheet recovery should contribute the preservations of cell phenotype and biological functions of cell sheet for applying to clinical trials.

Human Neural Tissue Construct Fabrication Based on Scaffold-Free Tissue Engineering.

Current neural tissue engineering strategies involve the development and application of neural tissue constructs produced by using an anisotropic polymeric scaffold. This study reports a scaffold-free method of tissue engineering to create a tubular neural tissue construct containing unidirectional neuron bundles. The surface patterning of a thermoresponsive culture substrate and a coculture system of neurons with patterned astrocytes can provide an anisotropic structure and easy handling of the neural tissue construct without the use of a scaffold. Furthermore, using a gelatin gel-coated plunger, the neuron bundles can be laid out in the same direction at regulated intervals within multilayered astrocyte sheets. Since the 3D tissue construct is composed only by neurons and astrocytes, they can communicate physiologically without obstruction of a scaffold. The medical benefits of scaffold-free tissue generation provide new opportunities for the development of human cell-based tissue models required to better understand the mechanisms of neurodegenerative diseases. Therefore, this new tissue engineering approach may be useful to establish a technology for regenerative medicine and drug discovery using the patients own neurons.

Controlling shape and position of vascular formation in engineered tissues by arbitrary assembly of endothelial cells.

Cellular self-assembly based on cell-to-cell communication is a well-known tissue organizing process in living bodies. Hence, integrating cellular self-assembly processes into tissue engineering is a promising approach to fabricate well-organized functional tissues. In this research, we investigated the capability of endothelial cells (ECs) to control shape and position of vascular formation using arbitral-assembling techniques in three-dimensional engineered tissues. To quantify the degree of migration of ECs in endothelial network formation, image correlation analysis was conducted. Positive correlation between the original positions of arbitrarily assembled ECs and the positions of formed endothelial networks indicated the potential for controlling shape and position of vascular formations in engineered tissues. To demonstrate the feasibility of controlling vascular formations, engineered tissues with vascular networks in triangle and circle patterns were made. The technique reported here employs cellular self-assembly for tissue engineering and is expected to provide fundamental beneficial methods to supply various functional tissues for drug screening and regenerative medicine.

The high functionalization of temperature-responsive culture dishes for establishing advanced cell sheet engineering

Our laboratory developed “cell sheet engineering”, which has been applied to regenerative medicine. However, the applications are restricted to the transplants of single-, double-, and triple-layered cell sheets, because of the poor supplies of nutrients and oxygen. To fabricate thicker and dense tissues in vitro, multi-layered cell sheets need to have capillary networks. Additionally, individual tissues found in animal bodies have individual specific micropatterns of cells for expressing their specific functions and characteristics. Therefore, our laboratory has developed various kinds of micropatterned surfaces which could align cells in the shape of micropatterns. In this review, we summarize and discuss patterned surfaces that allow us to perform the high functionalization of temperature-responsive culture dishes for establishing advanced cell sheet engineering.