研究者詳細 - 景山　達斗

Large-Scale Preparation of Hair Follicle Germs Using a Microfluidic Device

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：公益社団法人日本生物工学会共著

Ellen Sugiyama, Ayaka Nanmo, Xiaolei Nie, Shu-Yung Chang, Michinao Hashimoto, Atsushi Suzuki, Tatsu … 全著者表示

ACS Biomaterials Science & Engineering 10 ( 2 ) 998 - 1005 2024年1月 [査読有り]

Exosomes for hair growth and regeneration

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Chemical Society 単著

Hair follicle morphogenesis during embryonic development is driven by the formation of hair follicle germs (HFGs) via interactions between epithelial and mesenchymal cells. Bioengineered HFGs are potential tissue grafts for hair regenerative medicine because they can replicate interactions and hair follicle morphogenesis after transplantation. However, a mass preparation approach for HFGs is necessary for clinical applications, given that thousands of de novo hair follicles are required to improve the appearance of a single patient with alopecia. In this study, we developed a microfluidics-based approach for the large-scale preparation of HFGs. A simple flow-focusing microfluidic device allowed collagen solutions containing epithelial and mesenchymal cells to flow and generate collagen microbeads with distinct Janus structures. During the 3 days of culture, the collagen beads contracted owing to cellular traction forces, resulting in collagen- and cell-dense HFGs. The transplantation of HFGs into nude mice resulted in highly efficient de novo hair follicle regeneration. This method provides a scalable and robust tissue graft preparation approach for hair regeneration.

Yinghui Zhou, Jieun Seo, Shan Tu, Ayaka Nanmo, Tatsuto Kageyama, Junji Fukuda

Journal of Bioscience and Bioengineering 137 ( 1 ) 1 - 8 2024年1月 [査読有り]

Exosomes for hair growth and regeneration

記述言語：英語掲載種別：研究論文（学術雑誌）単著

Zhou Yinghui, Seo Jieun, Tu Shan, Nanmo Ayaka, Kageyama Tatsuto, Fukuda Junji

Journal of Bioscience and Bioengineering 137 ( 1 ) 1 - 8 2023年12月

Effects of oxytocin on the hair growth ability of dermal papilla cells

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Society for Biotechnology, Japan 共著

Exosomes are lipid bilayer vesicles, 30-200 nm in diameter, that are produced by cells and play essential roles in cell-cell communication. Exosomes have been studied in several medical fields including dermatology. Hair loss, a major disorder that affects people and sometimes causes mental stress, urgently requires more effective treatment. Because the growth and cycling of hair follicles are governed by interactions between hair follicle stem cells (HFSCs) and dermal papilla cells (DPCs), a better understanding of the mechanisms responsible for hair growth and cycling through exosomes may provide new insights into novel treatments for hair loss. In this review, we focused on the comprehensive knowledge and recent studies on exosomes in the field of hair development and regeneration. We classified exosomes of several cellular origins for the treatment of hair loss. Exosomes and their components, such as microRNAs, are promising drugs for effective hair loss treatment.

Tatsuto Kageyama, Jieun Seo, Lei Yan & Junji Fukuda

Scientific Reports 13 ( 1 ) 15587 2023年10月 [査読有り]

Cryopreservation of engineered hair follicle germs for hair regenerative medicine

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Portfolio 単著

Oxytocin (OXT) is a neuropeptide hormone termed "love hormone" produced and released during childbirth and lactation. It is also produced in response to skin stimulation (e.g., during hugging and massaging) and music therapy. The effects of OXT on various organs have been revealed in recent years; however, the relationship between hair follicles and OXT remains unclear. In this study, we examined the effects of OXT on dermal papilla (DP) cells that control hair growth by secreting growth/regression signals. Gene expression analysis revealed that DP signature markers were significantly upregulated in DP cells treated with OXT. In addition, we tested the hair growth-promoting effects of OXT using in vitro hair follicle organoids. OXT promoted the growth of hair peg-like sprouting by upregulating the expression of growth-promoting factors, including genes encoding vascular endothelial growth factor A (VEGFA). This study highlights the positive effects of OXT in hair follicles and may assist in the development of new treatments for alopecia.

Mio Aoki, Ryoto Yokota, Shoji Maruo, Tatsuto Kageyama, Junji Fukuda

Journal of Bioscience and Bioengineering 136 ( 3 ) 246 - 252 2023年9月 [査読有り]

Gelatin acrylamide with improved UV crosslinking and mechanical properties for 3D biofabrication

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Society for Biotechnology, Japan 単著

Hair regenerative medicine must involve practical procedures, such as cryopreservation of tissue grafts. This can aid in evaluating tissue safety and quality, as well as transportation to a clinic and multiple transplants. Hair follicle germs (HFGs), identified during in vivo development, are considered effective tissue grafts for hair regenerative medicine. However, to the best of our knowledge, methods for cryopreserving HFGs have not been explored yet. This study investigated the efficacy of slow vitrification methods for freezing HFGs. Cryoprotectants such as dimethyl sulfoxide (DMSO) and carboxylated poly-L-lysine were used for vitrification. The results indicate that DMSO vitrification yielded the most efficient de novo hair regeneration in mouse skin, comparable to that of non-cryoprotected HFGs. A microfinger was fabricated to scale up the cryopreservation method, considering that thousands of tissue grafts were required per patient in clinical practice. The microfinger can be used for a series of processes, holding the HFG, replacing it with a cryopreservation solution, freezing it in liquid nitrogen, thawing it in a warm medium, and transplanting it into the skin. Although de novo hair regeneration by HFGs cryopreserved using microfingers was reduced by approximately 20 % compared to those cryopreserved using flat plates for fertilized eggs, it exceeded 50 %. These findings demonstrate that vitrification with DMSO and microfingers could be a useful approach for the cryopreservation of tissue grafts in hair regenerative medicine for hair loss. (c) 2023, The Society for Biotechnology, Japan. All rights reserved.

Molino Binbin Zhang, O'Connell Cathal, Kageyama Tatsuto, Yan Lei, Wu Yumeng, Kawamura Izuru, Maruo … 全著者表示

Journal of Bioscience and Bioengineering 136 ( 1 ) 51 - 57 2023年6月

Bone beads enveloped with vascular endothelial cells for bone regenerative medicine *

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Society for Biotechnology, Japan 共著

Photocrosslinkable gelatin has attracted increasing interest in the field of biofabrication, with the most studied and widely used photocrosslinkable gelatin being gelatin methacrylate (GelMa). However, the 3D fabrication of GelMa has presented several limitations and challenges, primarily due to its slow crosslinking speed. It is generally known that acryl-based functional groups have faster reaction kinetics than methacryl-base groups. However, gelatin acrylamide (GelAc) has not been widely investigated, largely due to its increased complexity of synthesis relative to GelMA. In this study, we developed a novel synthesis method for GelAc. By varying the reaction ratio of reagents, GelAc with a degree of substitution from 20% to 95% was produced. The UV crosslinking properties of GelAc was studied, demonstrating significantly faster crosslinking kinetics than GelMa, especially at lower concentrations and low photoinitiator concentrations. The swelling ratio and mechanical properties of the crosslinked GelAc hydrogel were also characterized, and biocompatibility experiments conducted via both surface seeding and hydrogel encapsulation of cells, with good cell viability observed. The application of GelAc for 3D biofabrication was demonstrated by 3D printing. GelAc can be a useful material for the fabrication of 3D conduits for tissue engineering applications. & COPY; 2023, The Society for Biotechnology, Japan. All rights reserved.

Kageyama Tatsuto, Akieda Hikaru, Sonoyama Yukie, Sato Ken, Yoshikawa Hiroshi, Isono Hitoshi, Hirota … 全著者表示

Acta Biomaterialia 165 168 - 179 2023年6月

Gelatin acrylamide with improved UV crosslinking and mechanical properties for 3D biofabrication

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier Science Ltd. 共著

The transplantation of pre-vascularized bone grafts is a promising strategy to improve the efficacy of engraftment and bone regeneration. We propose a hydrogel microbead-based approach for preparing vascularized and high-density tissue grafts. Mesenchymal stem cell-encapsulated collagen microgels (2 & mu;L), termed bone beads, were prepared through spontaneous constriction, which improved the density of the mesenchymal stem cells and collagen molecules by more than 15-fold from the initial day of culture. Constriction was attributed to cell-attractive forces and involved better osteogenic differentiation of mesenchymal stem cells than that of spheroids. This approach was scalable, and & SIM;20 0 0 bone beads were prepared semi-automatically using a liquid dispenser and spinner flask. The mechanical stimuli in the spinner flask further improved the osteogenic differentiation of the mesenchymal stem cells in the bone beads compared with that in static culture. Vascular endothelial cells readily attach to and cover the surface of bone beads. The in vitro assembly of the endothelial cell-enveloped bone beads resulted in microchannel formation in the interspaces between the bone beads. Significant effects of endothelialization on in vivo bone regeneration were shown in rats with cranial bone defects. The use of endothelialized bone beads may be a scalable and robust approach for treating large bone defects. A unique aspect of this study is that the hMSC-encapsulated collagen microgels were prepared through spontaneous constriction, leading to the enrichment of collagen and cell density. This constriction resulted in favorable microenvironments for the osteogenic differentiation of hMSCs, which is superior to conventional spheroid culture. The microgel beads were then enveloped with vascular endothelial cells and assembled to fabricate a tissue graft with vasculature in the interspaces among the beads. The significant effects of endothelialization on in vivo bone regeneration were clearly demonstrated in rats with cranial bone defects. We believe that microgel beads covered with vascular endothelial cells provide a promising approach for engineering better tissue grafts for bone-regenerative medicine.& COPY; 2022 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY licens ( http://creativecommons.org/licenses/by/4.0/ )

Binbin Zhang Molino , Cathal O'Connell , Tatsuto Kageyama , Lei Yan , Yumeng Wu , Izuru Kawamu … 全著者表示

Journal of Bioscience and Bioengineering 136 ( 1 ) 51 - 57 2023年4月 [査読有り]

In vitro hair follicle growth model for drug testing

記述言語：英語掲載種別：研究論文（学術雑誌）単著

Tatsuto Kageyama , Hikaru Miyata , Jieun Seo , Ayaka Nanmo , Junji Fukuda

Scientific Reports 13 ( 1 ) 4847 2023年3月 [査読有り]

Expansion Culture of Hair Follicle Stem Cells through Uniform Aggregation in Microwell Array Devices

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Portfolio 単著

In vitro models of human hair follicle-like tissue could be fundamental tools to better understand hair follicle morphogenesis and hair drug screening. During prenatal development and postnatal cyclic hair regeneration, hair follicle morphogenesis is triggered by reciprocal interactions and the organization of the epithelial and mesenchymal cell populations. Given this mechanism, we developed an approach to induce hair peg-like sprouting in organoid cultures composed of epithelial and mesenchymal cells. Human fetal/adult epithelial and mesenchymal cells were cultured in a medium supplemented with a low concentration of either Matrigel or collagen I. These extracellular matrices significantly enhanced the self-organization capabilities of the epithelial and mesenchymal cells, resulting in spherical aggregation and subsequent hair peg-like sprouting. The length of the hair peg sprouting and associated gene expression significantly increased in the presence of a well-known hair drug, minoxidil. This approach may be beneficial for testing hair growth-promoting drug candidates.

Hirano Sugi, Kageyama Tatsuto, Yamanouchi Maki, Yan Lei, Suzuki Kohei, Ebisawa Katsumi, Kasai Keiic … 全著者表示

ACS BIOMATERIALS SCIENCE & ENGINEERING 9 ( 3 ) 1510 - 1519 2023年2月 [査読有り]

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Chemical Society 共著

Hair regeneration using hair follicle stem cells (HFSCs) and dermal papilla cells is a promising approach for the treatment of alopecia. One of the challenges faced in this approach is the quantitative expansion of HFSCs while maintaining their hair induction capacity. In this study, HFSC expansion was achieved through the formation of uniform-diameter cell aggregates that were subsequently encapsulated in Matrigel. We designed a microwell array device, wherein mouse HFSCs were seeded, allowed to form loosely packed aggregates for an hour, and then embedded in Matrigel. Quantitative analysis revealed a 20-fold increase in HFSC number in 2 weeks through this culture device. Gene expression of trichogenic stem cell markers in the device-grown cells showed a significant increase compared with that of typical flat substrate Matrigel suspension culture cells. These microwell array cultured HFSCs mixed with freshly isolated embryonic mesenchymal cells indicated vigorous hair regeneration on the skin of nude mice. Furthermore, we examined the feasibility of this approach for the expansion of human HFSCs from androgenetic alopecia patients and found that the ratio of CD200+ cells was improved significantly in comparison with that of cells cultured in a typical culture dish or in a Matrigel suspension culture on a flat substrate. Therefore, the novel approach proposed in this study may be useful for HFSC expansion in hair regenerative medicine.

毛髪再生医療の実現を目指して

景山達斗

生物工学会誌 101 ( 1 ) 38 - 38 2023年1月

Hypoxia inducible factor-1α promotes trichogenic gene expression in human dermal papilla cells

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：公益社団法人日本生物工学会単著

Jieun Seo, Lei Yan, Tatsuto Kageyama, Ayaka Nanmo, Yang-Sook Chun, Junji Fukuda

Scientific Reports 13 ( 1 ) 1478 2023年1月 [査読有り]

Impacts of manipulating cell sorting on in vitro hair follicle regeneration

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Portfolio 単著

Dermal papilla cells (DPCs) play critical roles in hair follicle development, but the underlying mechanisms that contribute to hair regeneration have yet to be fully elucidated, particularly in terms of alterations in androgenetic alopecia patients. In this study, we demonstrated that hypoxia-inducible factor-1 alpha (HIF-1 alpha) is suppressed in scalp tissues of androgenetic alopecia patients and potentially associated with hair follicle development. Using RT-qPCR and western blot, we found that mRNA and protein levels of trichogenic genes, LEF1 and versican (VCAN), were attenuated in HIF-1 alpha knockdown DPCs. Under an in vivo mimicked environment in a three-dimensional spheroid culture, HIF-1 alpha-suppressed DPCs downregulated the expression of hair induction-related genes. Finally, treatment with a HIF-1 alpha activator resulted in the elevated expression of trichogenic genes in DPCs. This study highlights the importance of dermal HIF-1 alpha expression in regulating trichogenic genes and provides a promising therapeutic target and a fundamental tissue engineering approach for hair loss treatment.

Kageyama Tatsuto, Anakama Riki, Togashi Hideru, Fukuda Junji

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 134 ( 6 ) 534 - 540 2022年12月 [査読有り]

Reprogramming of three-dimensional microenvironments for in vitro hair follicle induction

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Society for Biotechnology, Japan 共著

Hair follicle morphogenesis is triggered by epithelialemesenchymal interactions. Several approaches have been developed for preparing hair follicle organoids using epithelial and mesenchymal cells; however, the current understanding of the relevance of in vitro spontaneous organization processes to hair regeneration is limited. In the present study, we used Y27632, a rho-associated kinase inhibitor, to investigate the effects of manipulation of cell sorting on hair regeneration in vitro. Dissociated hair follicle-inducible epithelial and mesenchymal cells were cultured in Y27632containing media in 96-well plates or polydimethylsiloxane microarray plates. We found that Y27632 supplementation modulated the spatial distribution of epithelial and mesenchymal cells from a dumbbell shape to a coreeshell configuration via a spontaneous organization process. New hair follicles with typical morphological features emerged in the Y27632-treated core-shell-shaped aggregates, and hair shafts sprouted with approximately 100% efficiency in vitro. Gene chip analysis and pathway-inhibition experiments revealed that the phosphatidylinositol-3-kinase/protein kinase B- and Ras-signaling pathways were involved in hair-like sprouting in the Y27632-treated hair follicle organoids. Our findings enhance the understanding of hair follicle organogenesis and the development of hair follicle organoids. (c) 2022, The Society for Biotechnology, Japan. All rights reserved.

Kageyama Tatsuto, Shimizu Akihiro, Anakama Riki, Nakajima Rikuma, Suzuki Kohei, Okubo Yusuke, Fukud … 全著者表示

SCIENCE ADVANCES 8 ( 42 ) eadd4603 2022年10月 [査読有り]

Engineering hair follicle organoids through microenvironmental reprograming

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Association for the Advancement of Science (AAAS) 共著

During embryonic development, reciprocal interactions between epidermal and mesenchymal layers trigger hair follicle morphogenesis. This study revealed that microenvironmental reprogramming via control over these interactions enabled hair follicle induction in vitro. A key approach is to modulate spatial distributions of epithelial and mesenchymal cells in their spontaneous organization. The de novo hair follicles with typical morphological features emerged in aggregates of the two cell types, termed hair follicloids, and hair shafts sprouted with near 100% efficiency in vitro. The hair shaft length reached similar to 3 mm in culture. Typical trichogenic signaling pathways were up-regulated in hair follicloids. Owing to replication of hair follicle morphogenesis in vitro, melanosome production and transportation were also monitored in the hair bulb region. This in vitro hair follicle model might be valuable for better understanding hair follicle induction, evaluating hair growth and inhibition of hair growth by drugs, and modeling gray hairs in a well-defined environment.

Kageyama Tatsuto, Fukuda Junji

TISSUE ENGINEERING PART A 28 863 - 863 2022年10月

Web of Science

記述言語：日本語掲載種別：研究論文（学術雑誌）共著

Bone beads enveloped with vascular endothelial cells for bone regenerative medicine

Tatsuto Kageyama, Hikaru Akieda, Yukie Sonoyama, Ken Sato, Hiroshi Yoshikawa, Hitoshi Isono, Makot … 全著者表示

Acta Biomaterialia 165 168 - 179 2022年8月 [査読有り]

Effects of the PI3K/Akt signaling pathway on the hair inductivity of human dermal papilla cells in hair beads

記述言語：英語掲載種別：研究論文（学術雑誌）単著

Monami Yamane, Jieun Seo, Yinghui Zhou, Tomoki Asaba, Shan Tu, Ayaka Nanmo, Tatsuto Kageyama, Junji … 全著者表示

Journal of Bioscience and Bioengineering 134 ( 1 ) 55 - 61 2022年7月 [査読有り]

Cell-repellent polyampholyte for conformal coating on microstructures

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Society for Biotechnology, Japan 単著

Dermal papilla cells (DPCs), which play a central role in the regulation of hair follicle development and hair growth, are among the most promising cell sources for hair regenerative medicine. However, a critical issue in the use of DPCs is the immediate loss of hair inducing functions in typical two-dimensional (2D) culture. We have previously demonstrated that when DPCs are encapsulated in drops of collagen gel (named hair beads, HBs), the density of collagen and cells is concentrated >10-fold during 3 d of culture through the spontaneous constriction of the drops, leading to efficient hair follicle regeneration upon transplantation. However, the mechanisms responsible for the activation of the hair-inducing functions of DPCs have been poorly elucidated. Here, transcriptome comparisons of human DPCs in HB culture and in typical 2D culture revealed that the phosphoinositide 3-kinase and Akt (PI3K/Akt) signaling pathway was significantly upregulated in HB culture. Inhibition of the PI3K/Akt signaling pathway decreased the hair-inducing capability of DPCs in HBs, while the activation of the PI3K/Akt signaling pathway using an activator improved trichogenous gene expression of DPCs in 2D culture. These results suggest that the PI3K/Akt signaling pathway is crucial for the maintenance and restoration of hair inductivity of DPCs. HB culture and/or activators of the PI3K/Akt signaling pathway could be a promising strategy for preparing DPCs for hair regenerative medicine. (c) 2022, The Society for Biotechnology, Japan. All rights reserved.

Suzuki Kohei, Hiroi Yoshiomi, Abe-Fukasawa Natsuki, Nishino Taito, Shouji Takeaki, Katayama Junko, … 全著者表示

SCIENTIFIC REPORTS 12 ( 1 ) 2022年6月 [査読有り]

Luciferase assay system to monitor fibroblast growth factor signal disruption in human iPSCs

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Portfolio 共著

Repellent coatings are critical for the development of biomedical and analytical devices to prevent nonspecific protein and cell adhesion. In this study, prevelex (polyampholytes containing phosphate and amine units) was synthesized for the fine coating of microdevices for cell culture. The dip-coating of the prevelex on hydrophobic substrates altered their surfaces to be highly hydrophilic and electrically neutral. The range of prebake temperature (50-150 degrees C) after dip-coating was moderate and within a preferable range to treat typical materials for cell culture such as polystyrene and polydimethylsiloxane. Scanning electron microscopy revealed a conformal and ultra-thin film coating on the micro/nano structures. When compared with poly(2-hydroxyethyl methacrylate) and poly(2-methacryloyloxyethyl phosphorylcholine), prevelex exhibited better characteristics for coating on microwell array devices, thereby facilitating the formation of spheroids with uniform diameters using various cell types. Furthermore, to examine cellular functionalities, mouse embryonic epithelial and mesenchymal cells were seeded in a prevelex-coated microwell array device. The two types of cells formed hair follicle germ-like aggregates in the device. The aggregates were then transplanted to generate de novo hair follicles in nude mice. The coating material provided a robust and fine coating approach for the preparation of non-fouling surfaces for tissue engineering and biomedical applications.

Seiya Kanno, Kashu Mizota, Yusuke Okubo, Tatsuto Kageyama, Lei Yan, Junji Fukuda

STAR Protocols 3 ( 2 ) 101439 2022年6月 [査読有り]

Bioprinting of hair follicle germs for hair regenerative medicine

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier 単著

We describe a protocol for a live-cell luciferase assay system for continuously monitoring fibroblast growth factor (FGF) signal disruption in human-induced pluripotent stem cells (iPSCs). Signal disrupting effects of chemicals are used as an indicator to evaluate toxicity. The assay is reliably predictive of the effects of limb malformation chemicals (AUC = 0.93). The current approach is limited to FGF signal disruption, and combinations with other types of signaling will be required to detect the effects of different toxicants. For complete details on the use and execution of this protocol, please refer to Kanno et al. (2022a).

Ayaka Nanmo, Lei Yan, Tomoki Asaba, Licheng Wan, Tatsuto Kageyama, JunjiFukuda

Acta Biomaterialia 165 50 - 59 2022年6月 [査読有り]

ELECTRICAL STIMULATION VIA CONDUCTIVE POLYMER BED FOR HAIR FOLLICLE STEM CELL CULTURE

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier Science Ltd. 単著

Hair regenerative medicine is a promising approach to treat hair loss. The replication of in vivo tissue con-figurations and microenvironments, such as hair follicle germs, has been studied to prepare tissue grafts for hair regenerative medicine. However, such approaches should be scalable, because a single patient with alopecia requires thousands of tissue grafts. In this paper, we propose an approach for the scalable and automated preparation of highly hair-inductive tissue grafts using a bioprinter. Two collagen droplets (2 & mu;L each) containing mesenchymal and epithelial cells were placed adjacent to each other to fabricate hair-follicle-germ-like grafts. During three days of culture, the pairs of microgel beads were spontaneously contracted by cell traction forces, whereas the two cell types remained separated, where the densities of the cells and collagen were enriched more than 10 times. This approach allowed us to fabricate submil-limeter objects printed with millimeter-order accuracy, facilitating scalable and automated tissue graft preparation. Because of mesenchymal-epithelial interactions, hair microgels (HMGs, i.e., collagen-and cell-enriched microgels) efficiently regenerate hair follicles and shafts when transplanted into the back skin of mice. However, the generated hair shafts mostly remain under the skin. Therefore, we printed microgel beads onto surgical suture guides arrayed on a stage. The microgel beads were contracted along with the suture guides in culture prior to transplantation. The guide-inserted HMGs significantly im-proved hair-shaft sprouting through the skin, owing to the control of the orientation of the HMGs trans-planted into the skin. This approach is a promising strategy to advance hair regenerative medicine.Statement of significance This study proposes an approach for the scalable and automated preparation of highly hair-inductive grafts using a bioprinter. Two collagen droplets containing mesenchymal and epithelial cells were placed adjacently. Cell traction forces caused the pairs of microgel beads to spontaneously contract in culture. Because of mesenchymal-epithelial interactions, hair microgels (HMGs) efficiently regenerated hair fol-licles on the back skin of mice. However, the generated hair shafts remained mostly beneath the skin. Therefore, we printed microgel beads onto surgical suture guides arrayed on a stage. The guide-inserted HMGs significantly improved hair-shaft sprouting through the skin owing to the control of the orientation of the HMGs in the skin. This approach represents a promising strategy for advancing hair regenerative medicine.& COPY; 2022 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

Yan Lei, Kageyama Tatsuto, Zhang Binbin, Fukuda Junji

TISSUE ENGINEERING PART A 28 S310 - S310 2022年4月

Web of Science

記述言語：日本語掲載種別：研究論文（学術雑誌）共著

RESTORATION OF THE HAIR-INDUCTIVE CAPACITY OF HUMAN DERMAL PAPILLA CELLS EMBEDDED IN COLLAGEN MICROGEL

Yamane Monami, Kageyama Tatsuto, Fukuda Junji

TISSUE ENGINEERING PART A 28 S362 - S362 2022年4月

Web of Science

記述言語：日本語掲載種別：研究論文（学術雑誌）共著

Electrical stimulation, Dermal papilla cell, Polypyrrole, Tissue engineering, Hair regenerative medicine

Yan Lei, Kageyama Tatsuto, Zhang Binbin, Yamashita Seiya, Paul J. Molino, Gordon G. Wallace, Fukuda … 全著者表示

Journal of Bioscience and Bioengineering 133 ( 3 ) 281 - 290 2022年3月

Electrical stimulation to human dermal papilla cells for hair regenerative medicine

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Society for Biotechnology, Japan 共著

Hair follicle dermal papilla cells (DPCs) are specialized mesenchymal cells that play pivotal roles in hair formation, growth, and cycles, and they are considered as a cell source in hair regenerative medicine. Rodent dermal papilla cells have been shown to induce de novo hair follicle generation in the skin of recipients following transplantation, suggesting that dermal papilla cells can reprogram epidermal microenvironments. However, human DPCs (hDPCs) lose their ability to generate de novo hair follicles under conventional culture methods. We investigated the effects of electrical stimulation (ES) on hDPCs to restore the depressed trichogenic activity. We demonstrated that ES with a polypyrrole (PPy)-modified electrode upregulated trichogenic gene expression in hDPCs in vitro, and the activated cells when transplanted into mice generated double the number of hairs compared to that without the ES. Using specific inhibitors, we revealed that the mechanisms behind the electrical activation are associated with voltage-gated ion channels. Further, ES can be adapted for hDPCs from a patient with androgenic alopecia. Thus, this approach is potentially beneficial in preparing hDPCs for hair regenerative medicine.

Yan Lei, Kageyama Tatsuto, Zhang Binbin, Yamashita Seiya, Molino Paul J., Wallace Gordon G., Fukuda … 全著者表示

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 133 ( 3 ) 281 - 290 2022年3月 [査読有り]

Integrated fibroblast growth factor signal disruptions in human iPS cells for prediction of teratogenic toxicity of chemicals

記述言語：日本語掲載種別：研究論文（学術雑誌）共著

Kanno Seiya, Okubo Yusuke, Kageyama Tatsuto, Yan Lei, Fukuda Junji

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 133 ( 3 ) 291 - 299 2022年3月 [査読有り]

Establishment of a developmental toxicity assay based on human iPSC reporter to detect FGF signal disruption

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Society for Biotechnology, Japan 共著

The number of man-made chemicals has increased rapidly in recent decades, with certain chemicals potentially causing malformations in fetuses. Although the toxicities of chemicals have been tested in animals, chemicals that are not teratogenic in rodents can cause severe malformations in humans, owing to the differences in the susceptibility to the teratogenicity of chemicals among species. One possible cause of such species differences, other than pharmacokinetics, could be the difference in sensitivity to such chemicals at the cellular level. Therefore, a human cell-based high throughput assay system is needed for detecting potential teratogenic chemicals. In this study, we proposed a signal reporter assay using human induced pluripotent stem cells (iPSCs). Because developmental processes are governed by highly intricate and precisely programmed signaling pathways, external chemical-induced disruption of these pathways often triggers developmental toxicities. The reporter assay using hiPSCs was used to detect changes in the fibroblast growth factor (FGF) signaling pathway, a pathway essential for limb morphogenesis. The method was based on monitoring and time-accumulation of the signal disruption over time, rather than the classical endpoint detection of the signal disruption. This approach was useful for detecting signal disruptions caused by the malformation chemicals listed in the ICH S5 guideline, including thalidomide. The human iPSC-based signal disruption assay could be a promising tool for the initial screening of developmental toxicants. (c) 2021, The Society for Biotechnology, Japan. All rights reserved.

Kanno Seiya, Okubo Yusuke, Kageyama Tatsuto, Yan Lei, Kitajima Satoshi, Fukuda Junji

ISCIENCE 25 ( 2 ) 103770 2022年2月 [査読有り]

Optimized Microwell Array Device for Preparation of Hair Follicle Germ-like Aggregates

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell Press 共著

The number of man-made chemicals has increased exponentially recently, and exposure to some of them can induce fetal malformations. Because complex and precisely programmed signaling pathways play important roles in develop-mental processes, their disruption by external chemicals often triggers develop-mental toxicity. However, highly accurate and high-throughput screening assays for potential developmental toxicants are currently lacking. In this study, we pro-pose a reporter assay that utilizes human-induced pluripotent stem cells (iPSCs) to detect changes in fibroblast growth factor signaling, which is essential for limb morphogenesis. The dynamics of this signaling after exposure to a chemical were integrated to estimate the degree of signaling disruption, which afforded a good prediction of the capacity of chemicals listed in the ECVAM International Validation Study that induce limb malformations. This study presents an initial report of a human iPSC-based signaling disruption assay, which could be useful for the screening of potential developmental toxicants.

Suzuki Kohei, Hiroi Yoshiomi, Kageyama Tatsuto, Fukuda Junji

Alternatives to Animal Testing and Experimentation 27 ( 1 ) 14 - 23 2022年

Effects of Exosomes Derived from Dermal Papilla Cells on Hair Follicle Stem Cells and Hair Follicle Organoids

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：日本動物実験代替法学会共著

Hair follicle morphogenesis is triggered by reciprocal interactions between epithelial and mesenchymal layers in the hair follicle germ. Techniques to create follicle germ-like structures in vitro may be useful for understanding hair follicle morphogenesis and for creating transplantable tissue for hair regenerative medicine. Microwell arrays with oxygen-permeable materials have been proposed as a promising tool for in vitro fabrication of hair follicle germ. In this study, we investigated the effects of microwell morphology and culture process on the formation and function of hair follicle germ. Microwell geometries (flat, round-bottom, pen, and cone) significantly affected the formation of hair follicle germ and the expression of hair growth-related genes. Specifically, pen-type microwells enhanced the rate of follicle germ formation by promoting cell assembly. Even in typical round-bottom wells, centrifugal filling of cells into the microwells enhanced cell assembly and follicular germ formation rates. The higher the efficiency of two cell types to form two separate follicular germ, the higher the expression of hair growth-associated gene, versican. The highest expression of versican was observed when cells were centrifugally packed in round-bottom wells. Aside from hair follicle formation, these microwell array with various shapes may be useful for producing various other organoids and tissues.

Zhou Yinghui, Yamane Monami, Suzuki Kohei, Nanmo Ayaka, Tu Shan, Kageyama Tatsuto, Fukuda Junji

Alternatives to Animal Testing and Experimentation 27 ( 1 ) 1 - 13 2022年

Deep neural network for the determination of transformed foci in Bhas 42 cell transformation assay

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：日本動物実験代替法学会共著

Hair follicle development and cycle are regulated by the reciprocal interactions between epithelial and mesenchymal cells. Mechanisms responsible for the regulation have mainly been studied using experimental animals such as rodent fetuses. The purpose of this study is to understand such communications by using in vitro cell culture models of human hair follicle-derived cells. Human dermal papilla cells (DPCs) were cultured using 2-dimensional (2D), spheroid (3D), and spheroid microwell array cultures in an oxygen-permeable substrate (3D-oxy). Exosomes secreted in the culture media were collected and purified. All three types of exosomes moderately promoted proliferation of human hair follicle stem cells (HFSCs). Exosomes from the 3D-oxy culture most efficiently upregulated the expression of CD200, CD34, and K15 in HFSCs compared to those from the other two culture methods. To investigate the effects of exosomes on in vitro hair follicle formation, mouse embryonic epithelial and mesenchymal cells were used to prepare hair follicle organoids. The expression analysis of trichogenic and HFSC marker genes revealed that exosomes from the 3D-oxy culture were the most effective in maintaining stem cells and stimulating hair follicle sprouting in vitro. Exosomes from human DPCs may have substantial potential for treating patients with hair loss.

Masumoto Minami, Fukuda Ittetsu, Furihata Suguru, Arai Takahiro, Kageyama Tatsuto, Ohmori Kiyomi, S … 全著者表示

SCIENTIFIC REPORTS 11 ( 1 ) 23344 2021年12月 [査読有り]

3D-Printed Micro-Tweezers with a Compliant Mechanism Designed Using Topology Optimization

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Portfolio 共著

Bhas 42 cell transformation assay (CTA) has been used to estimate the carcinogenic potential of chemicals by exposing Bhas 42 cells to carcinogenic stimuli to form colonies, referred to as transformed foci, on the confluent monolayer. Transformed foci are classified and quantified by trained experts using morphological criteria. Although the assay has been certified by international validation studies and issued as a guidance document by OECD, this classification process is laborious, time consuming, and subjective. We propose using deep neural network to classify foci more rapidly and objectively. To obtain datasets, Bhas 42 CTA was conducted with a potent tumor promotor, 12-O-tetradecanoylphorbol-13-acetate, and focus images were classified by experts (1405 images in total). The labeled focus images were augmented with random image processing and used to train a convolutional neural network (CNN). The trained CNN exhibited an area under the curve score of 0.95 on a test dataset significantly outperforming conventional classifiers by beginners of focus judgment. The generalization performance of unknown chemicals was assessed by applying CNN to other tumor promotors exhibiting an area under the curve score of 0.87. The CNN-based approach could support the assay for carcinogenicity as a fundamental tool in focus scoring.

Yukihito Moritoki, Taichi Furukawa, Jinyi Sun, Minoru Yokoyama, Tomoyuki Shimono, Takayuki Yamada, … 全著者表示

Micromachines 12 ( 5 ) 579-1 - 579-10 2021年5月 [査読有り]

Impact of adipose-derived stem cells on engineering hair follicle germ-like tissue grafts for hair regenerative medicine

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI 共著

The development of handling technology for microscopic biological samples such as cells and spheroids has been required for the advancement of regenerative medicine and tissue engineering. In this study, we developed micro-tweezers with a compliant mechanism to manipulate organoids. The proposed method combines high-resolution microstereolithography that uses a blue laser and topology optimization for shape optimization of micro-tweezers. An actuation system was constructed using a linear motor stage with a force control system to operate the micro-tweezers. The deformation of the topology-optimized micro-tweezers was examined analytically and experimentally. The results verified that the displacement of the tweezer tip was proportional to the applied load; furthermore, the displacement was sufficient to grasp biological samples with an approximate diameter of several hundred micrometers. We experimentally demonstrated the manipulation of an organoid with a diameter of approximately 360 mu m using the proposed micro-tweezers. Thus, combining microstereolithography and topology optimization to fabricate micro-tweezers can be potentially used in modifying tools capable of handling various biological samples.

Rikuma Nakajima, Yoshiki Tate, Lei Yan, Tatsuto Kageyama, Junji Fukuda

Journal of bioscience and bioengineering 131 ( 6 ) 679 - 685 2021年3月 [査読有り]

Hair follicle germs containing vascular endothelial cells for hair regenerative medicine

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier 共著

Hair regenerative medicine has emerged as a promising treatment strategy for severe hair loss, such as end-stage androgenetic alopecia. Various approaches to engineering three-dimensional tissue grafts have been explored since they drive the ability to regenerate hair follicles when transplanted. In the present study, we demonstrated the assembly of human adipose-derived stem cells (hASCs) into hair follicle germ (HFG)-like aggregates for de novo hair regeneration. We mixed human dermal papilla cells (hDPCs), murine embryonic epithelial cells, and hASCs in suspension, and allowed them to form aggregates. During three days of culture, cells initially formed a single aggregate with a random distribution of the three cell types, but the epithelial and dermal papilla cells subsequently separated from each other and formed a dumbbell-shaped HFG, with hASCs localized on the hDPC aggregate side. The involvement of hASCs significantly increased gene expression associated with hair morphogenesis compared to HFGs without hASCs. The self-organization of the three cell types was observed in our scalable lab-made chip device. HFGs containing hASCs efficiently generated hair shafts upon transplantation to nude mice, while only a few shafts were generated with HFGs without hASCs. This approach may be a promising strategy for fabricating tissue grafts for hair regenerative medicine. (C) 2021, The Society for Biotechnology, Japan. All rights reserved.

Tatsuto Kageyama, Yang-Sook Chun, Junji Fukuda

Scientific reports 11 ( 1 ) 624-1 - 624-10 2021年1月 [査読有り]

Influence of the flow velocity on membrane-aerated biofilm reactors: Application of a rotating disk for local flow control

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Springer Nature 共著

Hair regenerative medicine has emerged as a promising approach for the treatment of severe hair loss. Recent advances in three-dimensional tissue engineering, such as formation of hair follicle germs (HFGs), have considerably improved hair regeneration after transplantation in animal models. Here, we proposed an approach for fabricating HFGs containing vascular endothelial cells. Epithelial, dermal papilla, and vascular endothelial cells initially formed a single aggregate, which subsequently became a dumbbell-shaped HFG, wherein the vascular endothelial cells localized in the region of dermal papilla cells. The HFGs containing vascular endothelial cells exhibited higher expression of hair morphogenesis-related genes in vitro, along with higher levels of hair shaft regeneration upon transplantation to the dorsal side of nude mice, than those without vascular endothelial cells. The generated hair follicles represented functional characteristics, such as piloerection, as well as morphological characteristics comparable to those of natural hair shafts. This approach may provide a promising strategy for fabricating tissue grafts with higher hair inductivity for hair regenerative medicine.

Tadashi Shoji, Ryuya Itoh, Tadashi Nittami, Tatsuto Kageyama, Miyuki Noguchi, AkihiroYamasaki

Biochemical engineering journal 164 107771-1 - 107771-12 2020年12月 [査読有り]

Effects of platelet-rich plasma on in vitro hair follicle germ preparation for hair regenerative medicine

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier 共著

Tatsuto Kageyama, Ayaka Nanmo, Lei Yan, Tadashi Nittami, Junji Fukuda

Journal of bioscience and bioengineering 130 ( 6 ) 666 - 671 2020年12月 [査読有り] [招待有り]

Additive Manufacturing of Micromanipulator Mounted on a Glass Capillary for Biological Applications

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier 共著

Kozaki Shingo, Moritoki Yukihito, Furukawa Taichi, Akieda Hikaru, Kageyama Tatsuto, Fukuda Junji, M … 全著者表示

MICROMACHINES 11 ( 2 ) 174 2020年2月 [査読有り]

マイクロ光造形による生体試料操作用マイクロマニピュレーション・ツールの開発

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI 共著

In this study, a three-dimensional (3D) micromanipulator mounted on a glass capillary is developed for handling biological samples, such as multicellular spheroids and embryos. To fabricate the micromanipulator, we developed an additive manufacturing system based on high-resolution microstereolithography using a 405-nm blue laser. The fabrication system makes it possible to fabricate 3D microstructures on a glass capillary with 2.5 mu m lateral resolution and 25 mu m layer thickness. We also demonstrated the capture and release of a spheroid with the micromanipulator fabricated using our additive manufacturing system. We showed that spheroids can be easily handled by a simple operation with minimal damage using a cage-like multiple finger structure. Additive manufacturing of tailor-made micromanipulators mounted on a glass capillary will be useful in biological and tissue engineering research.

盛一志仁, 狐崎進吾, 秋枝輝, 古川太一, 景山達斗, 福田淳二, 丸尾昭二

ロボティクス・メカトロニクス講演会講演概要集 2020 ( 0 ) 1P2 - N02 2020年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人日本機械学会共著

In this study, a three-dimensional (3D) micromanipulator mounted on a glass capillary is developed for handling biological samples such as spheroids and embryos. To fabricate the micromanipulator, we developed an additive manufacturing system based on high-resolution microstereolithography using a 405-nm blue laser. Using this fabrication system, the micromaniplulator mounted on a glass capillary was fabricated. We demonstrated the capture and release of a spheroid with the micromanipulator. It was confirmed that spheroids could be easily handled by a simple operation with minimal damage using a cage-like multiple finger structure. Additive manufacturing of tailor-made micromanipulators mounted on a glass capillary will be useful for biological and tissue engineering researches.

その他リンク： https://ci.nii.ac.jp/naid/130007943800

Preparation of hair beads and hair follicle germs for regenerative medicine

Kageyama Tatsuto, Yan Lei, Shimizu Akihiro, Maruo Shoji, Fukuda Junji

BIOMATERIALS 212 55 - 63 2019年8月 [査読有り]

記述言語：日本語掲載種別：研究論文（学術雑誌）共著

Tailored cell sheet engineering using microstereolithography and electrochemical cell transfer

Kobayashi Yuka, Cordonier Christopher E. J., Noda Yohei, Nagase Fuminori, Enomoto Junko, Kageyama T … 全著者表示

SCIENTIFIC REPORTS 9 2019年7月 [査読有り]

記述言語：日本語掲載種別：研究論文（学術雑誌）共著

Synergic effects of oxygen supply and antioxidants on pancreatic beta-cell spheroids

Myasnikova Dina, Osaki Tatsuya, Onishi Kisaki, Kageyama Tatsuto, Molino Binbin Zhang, Fukuda Junji

SCIENTIFIC REPORTS 9 2019年2月 [査読有り]

記述言語：日本語掲載種別：研究論文（学術雑誌）共著

Gold cleaning methods for preparation of cell culture surfaces for self-assembled monolayers of zwitterionic oligopeptides

Junko Enomoto, Tatsuto Kageyama, Dina Myasnikova, Kisaki Onishi, Yuka Kobayashi, Yoko Taruno, Takah … 全著者表示

Journal of Bioscience and Bioengineering 125 ( 5 ) 606 - 612 2018年5月 [査読有り]

DOI

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier 単著

Spontaneous hair follicle germ (HFG) formation in vitro, enabling the large-scale production of HFGs for regenerative medicine

Tatsuto Kageyama, Chisa Yoshimura, Dina Mysnikova, Ken Kataoka, Tadashi Nittami, Shoji Maruo, Junji … 全著者表示

Biomaterials 154 291 - 300 2018年2月 [査読有り]

記述言語：英語掲載種別：研究論文（学術雑誌）共著

Injectable Hydrogel with Slow Degradability Composed of Gelatin and Hyaluronic Acid Cross-Linked by Schiff’s Base Formation

Hozumi Takuro, Kageyama Tatsuto, Ohta Seiichi, Fukuda Junji, Ito Taichi

Biomacromolecules 19 ( 2 ) 288 - 297 2018年2月 [査読有り]

DOI

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier 共著

Flatbed epi relief-contrast cellular monitoring system for stable cell culture

Tatsuya Osaki, Tatsuto Kageyama, Yuka Shimazu, Dina Mysnikova, Shintaro Takahashi, Shinichi Takimot … 全著者表示

Scientific reports 7 1897 2017年5月 [査読有り]

記述言語：英語掲載種別：研究論文（学術雑誌）共著

Catch and release of target cells using aptamer-conjugated electroactive zwitterionic oligopeptide SAM

Junko Enomoto, Tatsuto Kageyama, Tatsuya Osaki, Flavia Bonalumi, Francesca Marchese, Alfonso Gautie … 全著者表示

Scientific reports 7 43375 2017年 [査読有り]

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP 共著

Engineering thick cell sheets by electrochemical desorption of oligopeptides on membrane substrates

Junko Enomoto, Naoto Mochizuki, Katsumi Ebisawa, Tatsuya Osaki, Tatsuto Kageyama, Dina Myasnikova, … 全著者表示

Regenerative Therapy 3 24 - 31 2016年 [査読有り]

記述言語：英語掲載種別：研究論文（学術雑誌）共著

In situ crosslinkable gelatin-CMC hydrogels designed for rapid engineering of perfusable vasculatures

Tatsuto Kageyama, Tatsuya Osaki, Junko Enomoto, Tadashi Nittami, Takuro Hozumi, Taichi Ito and Junj … 全著者表示

ACS Biomaterials Science & Engineering 2 ( 6 ) 1059 - 1066 2016年 [査読有り]

記述言語：英語掲載種別：研究論文（学術雑誌）共著

Acceleration of vascular sprouting from fabricated perfusable vascular-like structures

Tatsuya Osaki, Takahiro Kakegawa, Tatsuto Kageyama, Junko Enomoto, Tadashi Nittami and Junji Fukuda

Plos One 10 ( 4 ) e0123735 2015年 [査読有り]

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PUBLIC LIBRARY SCIENCE 共著

Rapid engineering of endothelial cell-lined vascular-like structures in in situ crosslinkable hydrogels

Tatsuto Kageyama, Takahiro Kakegawa, Tatsuya Osaki, Junko Enomoto, Taichi Ito, Tadashi Nittami and … 全著者表示

Biofabrication 6 ( 2 ) 025006 2014年 [査読有り]