基材の硬さとCuscuta japonica Choisyの複合効果による間葉系幹細胞の骨形成能の若返り。
キーワード：間葉系幹細胞, 老朽化, 基板剛性, 薬草, 骨形成性分化
2023.05～2024.06.

単一細胞分析を用いた、老化MSCの若返りを誘導する基質の硬さに関する研究
キーワード：MSCの老化、若返り、基質の硬さ、単一細胞分析
2019.07～2024.06.

長時間ライブセルイメージングを可能にするナノ粒子添加剤
キーワード：ナノ粒子添加剤, 長時間ライブセルイメージング
2019.06～2023.06.

機械的基質上の幹細胞におけるYAP / TAZ核ー細胞質シャトルリング
キーワード：YAP/TAZ, 核ー細胞質シャトルリング
2017.08～2020.08.

幹細胞のメカノトランスダクションとレドックス調節の相互作用
キーワード：メカノトランスダクション, ドックス
2015.08～2020.08.

「焦点接着タンパク質のライブセルイメージング」。
キーワード：ライブセルイメージング
2012.10～2014.12.

微視的培養弾性場の設計による幹細胞機能応答のメカノバイオロジー研究
キーワード：メカノバイオロジー
2008.04～2012.10.

「ソフトストライプパターン化ゲルを用いた細胞移動の方向制御」。
キーワード：ソフトストライプパターン化ゲル
2011.10～2014.01.

ダニ唾液腺における免疫調節タンパク質の特性解析とRNA干渉法によるダニの保護抗原の遺伝子抑制
キーワード：RNA干渉法
2006.10～2007.10.

Rejuvenation of mesenchymal stem cell osteogenic potency via the combined effects of substrate stiffness and Cuscuta japonica Choisy.
2023.05～2024.06, 代表者：Kuboki Thasaneeya, Kyushu University, JSPS (Japan)
This study aims to investigate the combined effects of substrate stiffness and Cuscuta japonica extract on osteogenic differentiation potency of the aged MSCs preconditioning on surface elasticity tunable hydrogels. The effect C. japonica extract on MSC senescence progression will be investigated by SA beta-GAL staining and real time PCR of cell cycle inhibitors and senescence markers. To evaluate the impact on osteogenic differentiation, the late passage MSCs will be passaging on tissue culture dish (TC) and gels Then, the osteogenic differentiation will be performed by culturing the cells that collected from TC and gels in the culture medium supply with various concentration of the seed extracts. The osteogenic differentiation potency will be investigated by staining of calcium deposition with Alizarin Red S, measurement of alkaline phosphatase activity and real time PCR of osteogenic differentiation markers. The substrate stiffness in combination with soluble factor C. japonica extract is expected to rescue therapeutic properties and promote the osteogenesis of the aged MSCs..

Measuring cell surface tension during metastasis.
2022.06～2024.06, 代表者：Sei Kuriyama, Akita University, Japan
This work aim to develop the fluorescent sensor probe for detecting the changes in cancer cell surface tension in respond to different substrate stiffness..

Understanding the effects of substrate stiffness on rejuvenation of aging MSC s via single cell analysis.
2019.07～2024.06, 代表者：Thasaneeya Kuboki, Kyushu University, Japan
Mesenchymal stem cells (MSCs) from bone marrow are widely used in clinical applications due to their therapeutic properties. However, in vitro expansion of MSCs on tissue culture dish (TC) induce aging, which reduce their quantity and qualities with undefined mechanism. This research aims to delineate the role substrate stiffness as potential modulator to delay the MSC aging process. The rejuvenation of the MSCs serially passaging on the engineered stiffness tunable gelatinous hydrogels will be evaluated. The innovative approach, single cell expression profiling, will be performed to gain a better understanding of mechano-regulation of MSC aging in single cell level for future development of biomaterials for MSC maintenance..

Application of nitroxide-radical nanoparticles for long-term time-lapse imaging.
2019.06～2023.06, 代表者：Thasaneeya Kuboki, Kyushu University, Japan
In this research, application of radical scavenger, nitroxide-radical nanoparticles (RNPs), during live cell imaging was investigated. In the fluorescent live cell imaging, addition of the RNPs could reduce the phototoxicity from the reactive oxygen species (ROS) that generated during time-lapse observation.

Elucidation of mechanical stimuli induced MSCs fate control via the expression of APC.
2017.06～2020.06, 代表者：Thasaneeya Kuboki, Kyushu University, Japan
Wnt signaling is one of the key players that control lineage specification in vertebrate embryo & pluripotency in embryonic stem cells (ES). In MSCs, various level of Wnt activity resulted in both inhibitory and stimulatory effects on MSC self-renewal and differentiation. A major effector of the canonical Wnt signaling pathway is the transcription factor β-catenin. Upon activation, β-catenin translocate into the nucleus and promote the expression of various genes. In absence of Wnt signal the destruction complex, formed by Adenomatous Polyposis Coli (APC), the scaffold Axin, and GSK3 β, which responsible for phosphorylation, ubiquitination and degradation of β-catenin via proteasome. APC is multifunctional protein that play pivotal role both in regulating canonical Wnt signaling pathway and cytoskeleton (CSK). APC proteins are essential negative regulators of Wnt signaling, however, the molecular connections between Wnt signaling, CSK dynamics and stem cell fate remain unclear. In our study, the expression of APC, was the highest and specifically up-regulated only on the patterned gels. Increased expression of APC in the frustration differentiation MSCs indicated the strong impact of mechanical stimuli that tightly regulate Wnt signaling. To gain insight into the precise role of APC in mechanotransduction of the MSCs, the expression level of APC in the MSCs that undergo differentiation and on the homogeneous or patterned gels will be analyzed in correlation with the stem cell differentiation/ stemness markers and effectors of Wnt signaling. .

Interplay between mechanotransduction and redox regulation of stem cells
2015.06～2018.06, 代表者：Thasaneeya Kuboki, Kyushu University, Japan
The cells are constantly exposed to several intracellular stresses such as reactive oxygen species (ROS) produced by oxidative metabolism and oxidative stress. ROS such as superoxide, hydroxyl radicals and hydrogen peroxide that mainly generated from NADPH oxidase from mitochondria could function as a second messenger in redox signaling. Some cells that localize in particular area also experience low oxygen condition (hypoxia) and develop an antioxidant defense system to maintain the oxidative/reductive (redox) homeostasis. For stem cells, the balance between ROS and antioxidants could regulate their fate, function and survival. Our previous proteomic analysis of MSCs on different elasticity gels revealed the differential expression of intracellular signaling molecules including the antioxidant enzymes. However, direct correlation between mechanotransduction and redox signaling has never been reported. Here, I investigate the interrelationships between these two distinct pathways. I observed the changes in expression of neurogenic markers in stem cells on the soft gels that mimic the brain (1 kPa) and osteogenic markers on the stiffer gels that mimic bone cells (80 kPa), together with differential expression of antioxidant genes. The mitochondria superoxide production of the cells was increased on the soft gels but suppressed on the stiff gels. These data indicated that the mechanical stimuli could modulate the cellular redox balance, ROS production and differentiation markers of the stem cells, reflecting the crosstalk between mechanotransduction and redox signaling..

Stem cell fate regulation on the mechanical engineered gelatinous gels.
2015.06～2020.06, 代表者：Thasaneeya Kuboki, Kyushu University, Japan
Evidences suggested that the stem cells have the imprint of the mechanical microenvironment that could interfere the cell plasticity and fate, indicating that the traditional rigid tissue culture dishes could bias the MSCs behaviors. In this study, the microelasticity-patterned gels (soft domain 3 kPa/stiff domain 30 kPa) were designed to induced the cells to move between the soft and stiffer regions with approximately equal residence time to reduce the mechanical memory and maintain the stemness. This MSCs defined as “frustration differentiation stem cells”. We cultured the MSCs on the control culture dishes, homogeneous soft (3 kPa), stiff (30 kPa) and the patterned gels for microarray analysis. The data suggested that the micropatterned gels could induced cellular movement, proliferation, inhibition of cell death and activation of signaling pathways related to stem cell pluripotency, indicating that the patterned gels provided the appropriate mechanical microenvironments for maintenance and expansion of stem cell. To gain deeper insight into the underlying mechanism, I’m investigating the dynamic of early event of mechanotransduction that regulate the cells behaviors. The nuclear translocation of key signaling components such as transcription factors (TFs) is an early and essential step in the control of gene expression by numerous extracellular signals. Many studies highlighted the important of cytoplasmic to nuclear shuttling of TFs as essential regulator of gene expression. YAP/TAZ are key signaling intermediates that link mechanical cues to MSC differentiation. I’m now studying the mechanism of how forces influence nuclear events by real time observation of YAP/TAZ nucleocytoplasmic shuttling in stem cells on mechanical substrate. .

Polarity generation in durotactic crawling cells just crossing an elasticity boundary
2015.08～2018.06, 代表者：Thasaneeya Kuboki, Kyushu University, Japan
To elucidate the mechanism of the generation of polarity to direct cell movement, the correlation between the macro- and microscopic aspects of cell polarization in durotaxis were investigated via a quantitative analysis of cell polarity and dynamics of the FA in durotactic motile cells on the patterned gels with a sharp transition between the soft (35 kPa) and stiff regions (300 kPa). By using the “persistence random deformation (PRD)” model that we recently established to explain the relationship of the cell movement and shape fluctuations, the analysis revealed that polarity is promptly generated as the cell approaches to the elasticity boundary. A Fluorescence Recovery After Photobleaching analysis showed the stiffness- and spatiotemporal-dependent dynamics of paxillin upon crossing the boundary. The mobile fractions of paxillin increased in the extended anterior part on the stiff region, corresponding with the enhancement of asymmetric shape fluctuation, magnitude of polarity and contraction-retraction movement during the first few hours. The results indicated that a gradual transition of dynamics in FAs found in the adhered interface of a single cell is potentially a prerequisite for the emergence of cell polarity and symmetry-breaking..

Durotaxis induced collective migration of neural crest cells
2015.06～2018.06, 代表者：Sei Kuriyama, Akita University, Japan
Collective cell migration is a fundamental process that enables the coordinated movement of groups of cells that remain connected via cell–cell junctions. Collective cell movements support the formation and morphological reshaping of larger tissue structures during the morphogenesis of ducts, glands, and vessels, as well as epithelial homeostasis and regeneration. In this study, durotaxis induced collective migration of the neural crest cells is being studied. We observed an interesting phenomenon that the microelasticity patterned gels could induce a pure durotactic collective migration of the NCs. We are now investigating the pathways responsible for this migration behaviors..

Fluorescence live cell imaging on nanosheets
2017.06～2018.06, 代表者：Kaoru Tamada, Kyushu University, Japan
In this collaborative research, high resolution imaging of the cells on the silver and gold nanosheets are being investigated. The nanosheets were fabricated using localized surface plasmon resonance (LSPR) technique. The LSPR of the silver/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. The genetic engineered cell lines that stably expressed focal adhesion protein paxillin were cultured on the nanosheet and live cell imaging showed high spatiotemporal resolution image. The construction of other fluorescence fusions/biosensor is now being performed for future application on dynamic observation of the proteins on the nanosheets. .

Redox gene expression of adipose-derived stem cells in response to soft hydrogel.
2013.08～2016.08, 代表者：Fahsai Kantawong, Chiang Mai University, Japan.

Time-dependent migratory behaviors in the long-term studies of fibroblast durotaxis on a hydrogel substrate fabricated with a soft band
2011.09～2014.01, 代表者：Kuboki Thasaneeya, Kyushu University, Kyushu University (Japan)
Long-term durotaxis of 3T3 fibroblasts was investigated on soft band gelatinous substrates. Most of the cell strongly repelled from the soft band on the first day of observation. Time-dependent migratory behaviors of the cells were observed during a time period of three days.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. .

2D-DIGE proteomic analysis of mesenchymal stem cell cultured on the elasticity-tunable hydrogels
2009.02～2011.09, 代表者：Kuboki Thasaneeya, Kyushu University, Kyushu University (Japan)
The proteomic profiles of stem cells cultured on different surface elasticity gelatin gels were studied using 2D-DIGE approach. Significant different in expression of several major cytoskeletal proteins and proteins related to important signalling cascades were observed on gel with different stiffness. .

主要原著論文

1.	Thasaneeya Kuboki, Hiroyuki Ebata, Tomoki Matsuda, Yoshiyuki Arai, Takeharu Nagai, and Satoru Kidoaki , Hierarchical development of motile polarity in durotactic cells just crossing an elasticity boundary, Cell Structure and Function, 2019.12.
2.	Kuboki T, Satoru Kidoaki, Fabrication of Elasticity-Tunable Gelatinous Gel for Mesenchymal Stem Cell Culture., Methods in Molecular Biology, 425-441, 2016.05, 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..
3.	Kantawong F, Kuboki T, Kidoaki S, Redox gene expression of adipose-Derived stem cells in response to soft hydrogel, Turkish Journal of Biology, 39, 682-691, 2015.08, Adipose-derived stem cells (ADSCs) showed morphological change to a neuron-like shape, and presented neuronal lineage bias when cultured on a very soft surface. To gain basic insight into gene expression relating to neuronal lineage bias in ADSCs, we examined the correlation between the gene expression levels of neuronal markers and redox proteins that were considered to have a close relation with lineage specification in stem cells. ADSCs were cultured on gelatinous soft hydrogel for 1-2 weeks. The time course changes in the expressions of neuronal genes (TUBB3 and NSE) and redox genes (TRX1, SOD1, SOD2, PRX2, GSTT1, and GSTP1) were monitored using real-time PCR. It was found that the TUBB3 gene had significantly upregulated compared to the control condition for tissue culture polystyrene, indicating that the neuronal gene expression of ADSCs could be achieved on soft hydrogel without the addition of any supplement. The expressions of the TRX1 and SOD1 genes were also observed to have significantly upregulated on the soft hydrogels. The results demonstrate that the upregulation of the neural marker of TUBB3 in the ADSCs correlates well with the upregulation of the redox genes of TRX1 and SOD1, when cultured on appropriate soft hydrogel substrates..
4.	Kuboki T, Chen W, Kidoaki S, Time-dependent migratory behaviors in the long-term studies of fibroblast durotaxis on a hydrogel substrate fabricated with a soft band., Langmuir, 10.1021/la501058j, 30, 6187-6196, 2014.05, 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..
5.	Kuboki T, Kantawong F, Burchmore R, Dalby MJ, Kidoaki S, 2D-DIGE proteomic analysis of mesenchymal stem cell cultured on the elasticity-tunable hydrogels., Cell Structure and Function, 37, 127-139, 2012.08, The present study focuses on mechanotransduction in mesenchymal stem cells (MSCs) in response to matrix elasticity. By using photocurable gelatinous gels with tunable stiffness, proteomic profiles of MSCs cultured on tissue culture plastic, soft (3 kPa) and stiff (52 kPa) matrices were deciphered using 2-dimensional differential in-gel analysis (2D-DIGE). The DIGE data, tied to immunofluorescence, indicated abundance and organization changes in the cytoskeletonal proteins as well as differential regulation of important signaling-related proteins, stress-responsing proteins and also proteins involved in collagen synthesis. The major CSK proteins including actin, tubulin and vimentin of the cells cultured on the gels were remarkably changed their expressions. Significant down-regulation of α-tubulin and β-actin can be observed on gel samples in comparison to the rigid tissue culture plates. The expression abundance of vimentin appeared to be highest in the MSCs cultured on hard gels. These results suggested that the substrate stiffness significantly affects expression balances in cytoskeletal proteins of MSCs with some implications to cellular tensegrity..
6.	Harnnoi T, Sakaguchi T, Nishikawa Y, Xuan X, Fujisaki K, Molecular characterization and comparative study of 6 salivary gland metalloproteases from the hard tick, Haemaphysalis longicornis. , Comp Biochem Physiol B Biochem Mol Biol, 147(1), 1, 93-101, 2007.05, Six genes encoding metalloproteases were identified from the salivary gland of the hard tick, Haemaphysalis longicornis. Comparative analyses have shown the evolutionary distinct and different mRNA expression patterns of each gene during blood feeding. The proteins are synthesized as proenzymes with a prodomain and a metalloprotease/cysteine-rich domain of the reprolysin family. Within the active site, amino acid substitutions were observed. The recombinant Escherichia coli expression of one gene, hlESTMP1, was performed. The immunoblot analysis and indirect fluorescent assay using anti-hlESTMP1 suggested that this protein is mainly expressed in the cytoplasm of the salivary glands and only the mature form of 34 kDa was detectable. The proenzyme expressed by baculovirus was processed into a mature domain, suggesting that proenzyme activation possibly occurs through a pro-protein convertase dependent pathway. The presence of these diverse enzymes might contribute to the greater functional complexity of bioactive molecules in tick saliva to facilitate blood feeding..
7.	Harnnoi T, Watchabunsook S, Sakaguchi T, Xuan X, Fujisaki K, Characterization of Haemaphysalis longicornis recombinant cement-like antigens and preliminary study of their vaccination effects., J Vet Med Sc, 68, 12, 1289-1295, 2006.12, Two genes encoding immunodominant antigens, hlim2 and hlim3, were obtained from a salivary gland cDNA library of the hard tick, Haemaphysalis longicornis. The recombinant proteins were expressed in Escherichia coli as the GST fusion protein and used for immunization. We observed that the attachment rate of nymphal ticks fed on mice immunized with GST-hlim3 was significantly lower than that in the control group during the initial days of feeding. However, immunization with GST-hlim3 did not affect the engorgement rate of the ticks. In sharp contrast, GST-hlim2 did not influence the attachment rate and feeding period of ticks but had a significant reduction in the engorgement body weight. These data highlight the suitability of the 2 recombinant cement-like proteins for use in a cocktail vaccine..
8.	Harnnoi T, Sakaguchi T, Xuan X, Fujisaki K, Identification of genes encoding cement-like antigens expressed in the salivary glands of Haemaphysalis longicornis. , J Vet Med Sci, 68, 11, 1155-1160, 2006.11, A cDNA expression library from the salivary glands of hard tick, Haemaphysalis longicornis, was constructed. Immunoscreening was performed using sera of the rabbit repeatedly infested with ticks and seventeen positive clones were obtained. A BLASTP search suggested that 8 sequences matched with that of hypothetical H. longicornis sequence and one clone encoded HL35 antigen U from the same tick species. Eight of 17 gave no match to any sequence reported in the database. The proteins expected from these novel sequences possess common characteristics with cement proteins which assist ticks in their attachment to the host during blood feeding. The expression of these genes in salivary glands was confirmed by RT-PCR. Four of the 8 sequences showed to be upregulated upon blood feeding. These immunodominant antigens are of particular interest as candidates for future cement protein based-tick vaccine..

主要学会発表等

1.	Thasaneeya Kuboki, Satoru Kidoaki, Elucidation of substrate stiffness mediated rejuvenation mechanism of aging stem cells through the mechano-regulation of cellular homeostasis, Molecular Biology Society of Japan, 2023.12.
2.	Thasaneeya Kuboki, Satoru Kidoaki, Elucidation of substrate stiffness mediated rejuvenation mechanism of aging stem cells through the regulation of cellular homeostasis, Japanese Biochemical Society, 2023.11.
3.	Thasaneeya KUBOKI, Satoru KIDOAKI, Understanding the mecahno-regulation of substrate stiffness on rejuvenation mechanism of aging stem cells, The Japan Society of Mechanical Engineers, 2022.11, Mesenchymal stem cells (MSCs) from bone marrow are widely used in clinical applications due to their therapeutic properties. However, in vitro expansion of MSCs on tissue culture dish (TC) induce aging (replicative senescence), which reduce their quantities and qualities with undefined mechanism. This research aims to delineate the role substrate stiffness as potential modulator to delay the MSC aging by elucidating the rejuvenation mechanism of the MSCs serial passaging on the engineered stiffness tunable gelatinous hydrogels. On TC, the cells remarkably changed their nuclear and cell morphology and exhibited the migration impairment. The later passage cells increased numbers of senescence associated-ß-Galactosidase positive cells, up-regulated the senescence markers and cell cycle inhibitors. On the other hand, the MSCs passaged on 3 and 20 kPa hydrogels could reverse these senescence phenotypes, as demonstrated by the decreasing the cell and nuclear size, increase motility and delayed senescence. The activation and de-activation of redox and antioxidant related molecules were also observed in the MSC passaged on the different surface elasticity substrates. These results indicated that the rigid TC potentially increase CSK tension and stress, while the hydrogel substrate could reverse these properties and rescue the cell functions, possibly through the mechano-regulation of CSK tension..
4.	Thasaneeya Kuboki, Satoru Kidoaki, Understanding the mecahno-regulation of substrate stiffness on rejuvenation mechanism of aging stem cells., Molecular Biology Society of Japan, 2022.11.
5.	Thasaneeya Kuboki, Satoru Kidoaki, Understanding the mecahno-regulation of substrate stiffness on rejuvenation mechanism of aging stem cell, world congress of biomechanics, 2022.07.
6.	Kuboki Thasaneeya, Kantawong Fahsai, Kidoaki Satoru, Effects of mechanical stimuli on redox homeostasis of mesenchymal stem cells, Office for the promotion of Gender equality, 2019.03.
7.	Kuboki Thasaneeya, Mechanotransduction and redox regulation of stem cells, Selectbio, 2018.07.
8.	Kuboki Thasaneeya, Kantawong Fahsai, Kidoaki Satoru, Mechanotransduction and redox regulation of stem cells., STEM18, 2018.03.
9.	THASANEEYA KUBOKI, Fahsai Kantawong, Satoru Kidoaki, Mechanotransduction and redox regulation of stem cells., Biomaterials International 2017 , 2017.08.
10.	THASANEEYA KUBOKI, Fahsai Kantawong, Satoru Kidoaki, Mechanotransduction and redox regulation of stem cells., ISB2017, 2017.07.
11.	THASANEEYA KUBOKI, Fahsai Kantawong, Satoru Kidoaki, Mechanotransduction and redox signaling of stem cells., Mechanobiology of Disease , 2016.09.
12.	THASANEEYA KUBOKI, Satoru Kidoaki, Live imaging of paxillin in durotactic migrating cells on the micro-elastically patterned hydrogels., KJF-ICOM , 2016.09.
13.	THASANEEYA KUBOKI, Satoru Kidoaki, Surface elasticity tunable gelatinous gel for manipulation of stem cell fate determination and directional cell migration., PCT, 2016.06.
14.	KUBOKI THASANEEYA, Japan Society of Mechanical Engineers 28; Bioengineering lecture , 2016.01, The mechanism of durotaxis was characterized using gelatinous square domain patterned gel on the soft base (300/40 kPa). The Fluorescence Recovery After Photobleaching analysis of 3T3 fiborblasts expressing venus-paxillin showed the stiffness, time and position dependent mobility of paxillin in the durotactic migrating cells. Time-lapse observation provided information concerning rate of assembly/disassembly and turnover of paxillin at the surface elasticity boundaries. .
15.	Kantawong F, Kuboki T, Kidoaki S, REDOX GENE EXPRESSION OF ADIPOSE-DERIVED STEM CELLS IN RESPONSE TO SOFT HYDROGEL, The 8th Asian-Pacific Conference on Biomechanics (AP-Biomech 2015), 2015.09, Introduction Adipose-derived stem cells (ADSCs) are multipotent stem cells within the adipose tissue, which are considered as a promising source of stem cell population. ADSCs offer unique opportunities as novel cell-based therapeutics and as traditional pharmaceutical discovery tools, which could have a significant therapeutic impact in the future. When cultured on very soft surface ADSCs showed morphological change to a neuron-like shape, and presented neuronal lineage bias. To gain the basic insights on gene expression relating to the neuronal lineage bias in ADSCs, we examined the correlation between the gene expression levels of neuronal markers and redox proteins that have recently been considered to have close relation with lineage specification in stem cells. Methods The surface elasticity tunable hydrogel was fabricated using photocurable styrenated gelatin. The elasticity of the gelatinous gel was measured using atomic force microscope and the 4 kPa hydrogels were used for ADSCs cultured for 4 days to 2 weeks. The time course expression of neuronal genes (TUBB3 and NSE) and redox genes (TRX1, SOD1, SOD2, PRX2, GSTT1, and GSTP1) were monitored using real-time PCR. Result and discussion It was found that the TUBB3 gene had significantly up-regulated, compared with the control tissue culture polystyrene, indicating that the neuronal gene expression of ADSCs could be achieved on soft hydrogel without the addition of any supplement. The expressions of the TRX1 and SOD1 genes were also observed to have significantly up-regulated on the soft hydrogels. The results demonstrate that the up-regulation of the neural marker of TUBB3 in the ADSCs correlates well with the up-regulation of the redox genes of TRX1 and SOD1 when cultured on appropriate soft hydrogel substrates. The changes in expression of these redox genes might have roles to play in the expression of neuronal markers, or they could be a response to the oxidative stress caused by the hydrogels, regarding which further studies are required. .
16.	THASANEEYA KUBOKI, Time-dependent migratory behaviors in the long-term studies of fibroblast durotaxis on a hydrogel substrate fabricated with a soft band , Mechanobiology, 2014.05, 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 µm, 50 µm, 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 three days. During the first day, most of the cells were strongly repelled by the soft band independent of band-width, 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. .
17.	Kuboki T, Chen W, Kidoaki S, Controlling mechano-repellent cell migration induced around a micro-scale soft stripe on hydrogel matrix. , International Nanomedicine Conference, (Sydney, Australia), 2013.07, Cell migration is a fundamental aspect of many physiological and pathological processes such as embryonic development, tissue morphogenesis, wound healing and cancer metastasis. Various factors in cellular microenvironment participate in the regulation of cell migration including soluble factors and mechanical stimuli from extracellular matrix. Fabrication of mechanically patterned substrates is essential in the understanding of how cell migration is affected by mechanical cues. Mechanotaxis or durotaxis describes the phenomenon that cells preferentially migrate toward stiffer domains on a substrate where a mechanical gradient is present 1. Our study focused on surface elasticity-induced directional cell migration. We demonstrated the feasibility in controlling directional cell migration, i.e. turning or repelling, based on mechanotaxis using patterned gels containing a single soft stripe (Fig. 1). The photocurable styrenated gelatin 2, 3, was used for the fabrication of patterned gels containing a narrow soft stripe (20, 50 or 150 µm) against a stiffer background, which was prepared using a newly developed Liquid Crystal Display (LCD) projector photolithographic patterning method 4. The surface elasticity of the hard domains (400 kPa) and the soft stripes (80 kPa) were designed according to the condition required to induce mechanotaxis 3. Repellency of 3T3 fibroblasts migrating from the hard domain by the soft stripe was observed in a time-dependent manner. The narrowest 20-µm wide stripes induced the strongest cell repellency, and similar trends were observed in conditions of stripe with three different widths. During the first day of time-lapse observations, majority of the cells were repelled upon approaching the soft regions. After the second and third days, fewer cells were repelled and more cells managed to cross the soft stripes. No noticeable changes in surface topography and gel elasticity after prolong cell cultured were observed. It was speculated that the time-dependent cell migratory behaviours were attributed to the secreted collagen deposited over time at the elasticity boundaries, as the fibroblasts are known to produce collagen. Immunofluorescense staining with anti-collagen I indicated that collagen deposition is a likely contributor to the changes in the cell repellency by the soft stripes over the time. .

特許出願・取得

海外渡航状況, 海外での教育研究歴