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Fumio Arai Last modified date：2024.04.11

Professor / Department of Stem Cell Biology and Medicine / Department of Stem Cell Biology and Medicine / Faculty of Medical Sciences


Papers
1. Kentaro Nakashima, Yuya Kunisaki, Kentaro Hosokawa, Kazuhito Gotoh, Hisayuki Yao, Ryosuke Yuta, Yuichiro Semba, Jumpei Nogami, Yoshikane Kikushige, Patrick S Stumpf, Ben D MacArthur, Dongchon Kang, Koichi Akashi, Shouichi Ohga, Fumio Arai, POT1a deficiency in mesenchymal niches perturbs B-lymphopoiesis., Communications biology, 10.1038/s42003-023-05374-0, 6, 1, 996-996, 2023.09, Protection of telomeres 1a (POT1a) is a telomere binding protein. A decrease of POT1a is related to myeloid-skewed haematopoiesis with ageing, suggesting that protection of telomeres is essential to sustain multi-potency. Since mesenchymal stem cells (MSCs) are a constituent of the hematopoietic niche in bone marrow, their dysfunction is associated with haematopoietic failure. However, the importance of telomere protection in MSCs has yet to be elucidated. Here, we show that genetic deletion of POT1a in MSCs leads to intracellular accumulation of fatty acids and excessive ROS and DNA damage, resulting in impaired osteogenic-differentiation. Furthermore, MSC-specific POT1a deficient mice exhibited skeletal retardation due to reduction of IL-7 producing bone lining osteoblasts. Single-cell gene expression profiling of bone marrow from POT1a deficient mice revealed that B-lymphopoiesis was selectively impaired. These results demonstrate that bone marrow microenvironments composed of POT1a deficient MSCs fail to support B-lymphopoiesis, which may underpin age-related myeloid-bias in haematopoiesis..
2. Keiyo Takubo, Phyo Wai Htun, Takeshi Ueda, Yasuyuki Sera, Masayuki Iwasaki, Miho Koizumi, Kohei Shiroshita, Hiroshi Kobayashi, Miho Haraguchi, Shintaro Watanuki, Zen-Ichiro Honda, Norimasa Yamasaki, Ayako Nakamura-Ishizu, Fumio Arai, Noboru Motoyama, Tomohisa Hatta, Tohru Natsume, Toshio Suda, Hiroaki Honda, MBTD1 preserves adult hematopoietic stem cell pool size and function., Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.2206860120, 120, 32, e2206860120, 2023.08, Mbtd1 (mbt domain containing 1) encodes a nuclear protein containing a zinc finger domain and four malignant brain tumor (MBT) repeats. We previously generated Mbtd1-deficient mice and found that MBTD1 is highly expressed in fetal hematopoietic stem cells (HSCs) and sustains the number and function of fetal HSCs. However, since Mbtd1-deficient mice die soon after birth possibly due to skeletal abnormalities, its role in adult hematopoiesis remains unclear. To address this issue, we generated Mbtd1 conditional knockout mice and analyzed adult hematopoietic tissues deficient in Mbtd1. We observed that the numbers of HSCs and progenitors increased and Mbtd1-deficient HSCs exhibited hyperactive cell cycle, resulting in a defective response to exogenous stresses. Mechanistically, we found that MBTD1 directly binds to the promoter region of FoxO3a, encoding a forkhead protein essential for HSC quiescence, and interacts with components of TIP60 chromatin remodeling complex and other proteins involved in HSC and other stem cell functions. Restoration of FOXO3a activity in Mbtd1-deficient HSCs in vivo rescued cell cycle and pool size abnormalities. These findings indicate that MBTD1 is a critical regulator for HSC pool size and function, mainly through the maintenance of cell cycle quiescence by FOXO3a..
3. Sahoko Matsuoka, Raffaella Facchini, Tiago C Luis, Joana Carrelha, Petter S Woll, Takuo Mizukami, Bishan Wu, Hanane Boukarabila, Mario Buono, Ruggiero Norfo, Fumio Arai, Toshio Suda, Adam Mead, Claus Nerlov, Sten Eirik W Jacobsen, Loss of endothelial membrane KIT Ligand affects systemic KIT ligand levels but not bone marrow hematopoietic stem cells., Blood, 10.1182/blood.2022019018, 2023.08, A critical regulatory role of hematopoietic stem cell vascular niches in the bone marrow has been implicated to occur through endothelial niche cell expression of KIT Ligand. However, endothelial-derived KIT Ligand is expressed in both a soluble and membrane-bound form, and not unique to bone marrow niches and is also systemically distributed through the circulatory system. Here we confirm that upon deletion of both the soluble and membrane-bound form of endothelial-derived KIT Ligand hematopoietic stem cells are reduced in mouse bone marrow. However, deletion of endothelial-derived KIT Ligand was also accompanied by reduced soluble KIT Ligand levels in blood, precluding any conclusion as to whether the reduction in HSC numbers reflect reduced endothelial expression of KIT Ligand within HSC niches, elsewhere in the bone marrow and/or systemic sKIT Ligand produced by endothelial cells outside of the bone marrow. Notably, endothelial deletion specifically of the membrane bound form of KIT Ligand also reduced systemic levels of soluble KIT Ligand although with no effect on stem cell numbers, implicating a hematopoietic stem cell regulatory role primarily of soluble rather than membrane KIT Ligand expression in endothelial cells. In support of a role of systemic rather than local niche expression of soluble KIT Ligand, hematopoietic stem cells were unaffected in bones with deletion of KIT ligand when implanted in mice with normal systemic levels of soluble KIT Ligand. Our findings highlight the need for more specific tools to unravel niche-specific roles of regulatory cues expressed in hematopoietic niche cells in the bone marrow..
4. Yaeko Nakajima-Takagi, Motohiko Oshima, Junichiro Takano, Shuhei Koide, Naoki Itokawa, Shun Uemura, Masayuki Yamashita, Shohei Andoh, Kazumasa Aoyama, Yusuke Isshiki, Daisuke Shinoda, Atsunori Saraya, Fumio Arai, Kiyoshi Yamaguchi, Yoichi Furukawa, Haruhiko Koseki, Tomokatsu Ikawa, Atsushi Iwama, Polycomb repressive complex 1.1 coordinates homeostatic and emergency myelopoiesis., eLife, 10.7554/eLife.83004, 12, 2023.06, Polycomb repressive complex (PRC) 1 regulates stem cell fate by mediating mono-ubiquitination of histone H2A at lysine 119. While canonical PRC1 is critical for hematopoietic stem and progenitor cell (HSPC) maintenance, the role of non-canonical PRC1 in hematopoiesis remains elusive. PRC1.1, a non-canonical PRC1, consists of PCGF1, RING1B, KDM2B, and BCOR. We recently showed that PRC1.1 insufficiency induced by the loss of PCGF1 or BCOR causes myeloid-biased hematopoiesis and promotes transformation of hematopoietic cells in mice. Here we show that PRC1.1 serves as an epigenetic switch that coordinates homeostatic and emergency hematopoiesis. PRC1.1 maintains balanced output of steady-state hematopoiesis by restricting C/EBPa-dependent precocious myeloid differentiation of HSPCs and the HOXA9- and β-catenin-driven self-renewing network in myeloid progenitors. Upon regeneration, PRC1.1 is transiently inhibited to facilitate formation of granulocyte-macrophage progenitor (GMP) clusters, thereby promoting emergency myelopoiesis. Moreover, constitutive inactivation of PRC1.1 results in unchecked expansion of GMPs and eventual transformation. Collectively, our results define PRC1.1 as a novel critical regulator of emergency myelopoiesis, dysregulation of which leads to myeloid transformation..
5. Kenta Murakami, Nobuhiko Hamazaki, Norio Hamada, Go Nagamatsu, Ikuhiro Okamoto, Hiroshi Ohta, Yoshiaki Nosaka, Yukiko Ishikura, Tomoya S Kitajima, Yuichiro Semba, Yuya Kunisaki, Fumio Arai, Koichi Akashi, Mitinori Saitou, Kiyoko Kato, Katsuhiko Hayashi, Generation of functional oocytes from male mice in vitro., Nature, 10.1038/s41586-023-05834-x, 615, 7954, 900-906, 2023.03, Sex chromosome disorders severely compromise gametogenesis in both males and females. In oogenesis, the presence of an additional Y chromosome or the loss of an X chromosome disturbs the robust production of oocytes1-5. Here we efficiently converted the XY chromosome set to XX without an additional Y chromosome in mouse pluripotent stem (PS) cells. In addition, this chromosomal alteration successfully eradicated trisomy 16, a model of Down's syndrome, in PS cells. Artificially produced euploid XX PS cells differentiated into mature oocytes in culture with similar efficiency to native XX PS cells. Using this method, we differentiated induced pluripotent stem cells from the tail of a sexually mature male mouse into fully potent oocytes, which gave rise to offspring after fertilization. This study provides insights that could ameliorate infertility caused by sex chromosome or autosomal disorders, and opens the possibility of bipaternal reproduction..
6. Yasutaka Hayashi, Kimihito C Kawabata, Yosuke Tanaka, Yasufumi Uehara, Yo Mabuchi, Koichi Murakami, Akira Nishiyama, Shigeru Kiryu, Yusuke Yoshioka, Yasunori Ota, Tatsuki Sugiyama, Keiko Mikami, Moe Tamura, Tsuyoshi Fukushima, Shuhei Asada, Reina Takeda, Yuya Kunisaki, Tomofusa Fukuyama, Kazuaki Yokoyama, Tomoyuki Uchida, Masao Hagihara, Nobuhiro Ohno, Kensuke Usuki, Arinobu Tojo, Yoshio Katayama, Susumu Goyama, Fumio Arai, Tomohiko Tamura, Takashi Nagasawa, Takahiro Ochiya, Daichi Inoue, Toshio Kitamura, MDS cells impair osteolineage differentiation of MSCs via extracellular vesicles to suppress normal hematopoiesis., Cell reports, 10.1016/j.celrep.2022.110805, 39, 6, 110805-110805, 2022.05, Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis and frequent progression to leukemia. It has long remained unresolved how MDS cells, which are less proliferative, inhibit normal hematopoiesis and eventually dominate the bone marrow space. Despite several studies implicating mesenchymal stromal or stem cells (MSCs), a principal component of the HSC niche, in the inhibition of normal hematopoiesis, the molecular mechanisms underlying this process remain unclear. Here, we demonstrate that both human and mouse MDS cells perturb bone metabolism by suppressing the osteolineage differentiation of MSCs, which impairs the ability of MSCs to support normal HSCs. Enforced MSC differentiation rescues the suppressed normal hematopoiesis in both in vivo and in vitro MDS models. Intriguingly, the suppression effect is reversible and mediated by extracellular vesicles (EVs) derived from MDS cells. These findings shed light on the novel MDS EV-MSC axis in ineffective hematopoiesis..
7. Katsuhiro Konno, Jingjing Kobayashi-Sun, Fumio Arai, Isao Kobayashi, Daisuke Sugiyama, Hematopoietic Cell Isolation by Antibody-Free Flow Cytometry in the Zebrafish Embryo, Methods in molecular biology, 10.1007/7651_2021_459, 2520, 171-180, 2022.05.
8. Patrick S Stumpf, Fumio Arai, Ben D MacArthur, Modeling Stem Cell Fates using Non-Markov Processes., Cell stem cell, 10.1016/j.stem.2021.01.009, 28, 2, 187-190, 2021.02, Epigenetic memories play an important part in regulating stem cell identities. Tools from the theory of non-Markov processes may help us understand these memories better and develop a more integrated view of stem cell fate and function..
9. Patrick S Stumpf, Xin Du, Haruka Imanishi, Yuya Kunisaki, Yuichiro Semba, Timothy Noble, Rosanna C G Smith, Matthew Rose-Zerili, Jonathan J West, Richard O C Oreffo, Katayoun Farrahi, Mahesan Niranjan, Koichi Akashi, Fumio Arai, Ben D MacArthur, Transfer learning efficiently maps bone marrow cell types from mouse to human using single-cell RNA sequencing., Communications biology, 10.1038/s42003-020-01463-6, 3, 1, 736-736, 2020.12, Biomedical research often involves conducting experiments on model organisms in the anticipation that the biology learnt will transfer to humans. Previous comparative studies of mouse and human tissues were limited by the use of bulk-cell material. Here we show that transfer learning-the branch of machine learning that concerns passing information from one domain to another-can be used to efficiently map bone marrow biology between species, using data obtained from single-cell RNA sequencing. We first trained a multiclass logistic regression model to recognize different cell types in mouse bone marrow achieving equivalent performance to more complex artificial neural networks. Furthermore, it was able to identify individual human bone marrow cells with 83% overall accuracy. However, some human cell types were not easily identified, indicating important differences in biology. When re-training the mouse classifier using data from human, less than 10 human cells of a given type were needed to accurately learn its representation. In some cases, human cell identities could be inferred directly from the mouse classifier via zero-shot learning. These results show how simple machine learning models can be used to reconstruct complex biology from limited data, with broad implications for biomedical research..
10. Katsuhiro Konno, Kasem Kulkeaw, Manabu Sasada, Takenobu Nii, Ayako Kaneyuki, Tohru Ishitani, Fumio Arai, Daisuke Sugiyama, A novel method to purify neutrophils enables functional analysis of zebrafish hematopoiesis., Genes to cells : devoted to molecular & cellular mechanisms, 10.1111/gtc.12810, 25, 12, 770-781, 2020.12, Zebrafish is a useful model to study vertebrate hematopoiesis, but lack of antibodies to zebrafish proteins has limited purification of hematopoietic cells. Here, we purified neutrophils from larval and adult zebrafish using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5, a DNA-staining fluorescent dye. In adult kidney marrow, we purified neutrophil-like PHA-E4low DRAQ5low cells, which neutrophil-type granules. Specifically, at 96-hr post-fertilization, we sorted large-sized cells from larvae using forward scatter and found that they consisted of PHA-Elow DRAQ5low populations. These cells had myeloperoxidase activity, were Sudan Black B-positive and expressed high levels of neutrophil-specific (csf3r and mpx) mRNAs, all neutrophil characteristics. Using this method, we conducted functional analysis suggesting that zyxin (Zyx) plays a role in neutrophil generation in zebrafish larvae. Overall, PHA-E and DRAQ5-based flow cytometry serves as a tool to purify zebrafish neutrophils..
11. Fumio Arai, Patrick S Stumpf, Yoshiko M Ikushima, Kentaro Hosokawa, Aline Roch, Matthias P Lutolf, Toshio Suda, Ben D MacArthur, Machine Learning of Hematopoietic Stem Cell Divisions from Paired Daughter Cell Expression Profiles Reveals Effects of Aging on Self-Renewal., Cell systems, 10.1016/j.cels.2020.11.004, 11, 6, 640-652, 2020.12, Changes in stem cell activity may underpin aging. However, these changes are not completely understood. Here, we combined single-cell profiling with machine learning and in vivo functional studies to explore how hematopoietic stem cell (HSC) divisions patterns evolve with age. We first trained an artificial neural network (ANN) to accurately identify cell types in the hematopoietic hierarchy and predict their age from single-cell gene-expression patterns. We then used this ANN to compare identities of daughter cells immediately after HSC divisions and found that the self-renewal ability of individual HSCs declines with age. Furthermore, while HSC cell divisions are deterministic and intrinsically regulated in young and old age, they are variable and niche sensitive in mid-life. These results indicate that the balance between intrinsic and extrinsic regulation of stem cell activity alters substantially with age and help explain why stem cell numbers increase through life, yet regenerative potency declines..
12. Patrick S Stumpf, Fumio Arai, Ben D MacArthur, Heterogeneity and 'memory' in stem cell populations., Physical biology, 10.1088/1478-3975/abba85, 17, 6, 065013-065013, 2020.11, Modern single cell experiments have revealed unexpected heterogeneity in apparently functionally 'pure' cell populations. However, we are still lacking a conceptual framework to understand this heterogeneity. Here, we propose that cellular memories-changes in the molecular status of a cell in response to a stimulus, that modify the ability of the cell to respond to future stimuli-are an essential ingredient in any such theory. We illustrate this idea by considering a simple age-structured model of stem cell proliferation that takes account of mitotic memories. Using this model we argue that asynchronous mitosis generates heterogeneity that is central to stem cell population function. This model naturally explains why stem cell numbers increase through life, yet regenerative potency simultaneously declines..
13. Kazuhito Gotoh, Yuya Kunisaki, Soichi Mizuguchi, Daiki Setoyama, Kentaro Hosokawa, Hisayuki Yao, Yuya Nakashima, Mikako Yagi, Takeshi Uchiumi, Yuichiro Semba, Jumpei Nogami, Koichi Akashi, Fumio Arai, Dongchon Kang, Mitochondrial Protein Synthesis Is Essential for Terminal Differentiation of CD45- TER119-Erythroid and Lymphoid Progenitors., iScience, 10.1016/j.isci.2020.101654, 23, 11, 101654-101654, 2020.11, p32/C1qbp regulates mitochondrial protein synthesis and is essential for oxidative phosphorylation in mitochondria. Although dysfunction of p32/C1qbp impairs fetal development and immune responses, its role in hematopoietic differentiation remains unclear. Here, we found that mitochondrial dysfunction affected terminal differentiation of newly identified erythroid/B-lymphoid progenitors among CD45- Ter119- CD31- triple-negative cells (TNCs) in bone marrow. Hematopoietic cell-specific genetic deletion of p32/C1qbp (p32cKO) in mice caused anemia and B-lymphopenia without reduction of hematopoietic stem/progenitor cells. In addition, p32cKO mice were susceptible to hematopoietic stress with delayed recovery from anemia. p32/C1qbp-deficient CD51- TNCs exhibited impaired mitochondrial oxidation that consequently led to inactivation of mTORC1 signaling, which is essential for erythropoiesis. These findings uncover the importance of mitochondria, especially at the stage of TNCs during erythropoiesis, suggesting that dysregulation of mitochondrial protein synthesis is a cause of anemia and B-lymphopenia with an unknown pathology..
14. Yasutaka Hayashi, Kimihito Cojin Kawabata, Yosuke Tanaka, Yasufumi Uehara, Shigeru Kiryu, Yasunori Ota, Yusuke Yoshioka, Yo Mabuchi, Tatsuki Sugiyama, Keiko Mikami, Moe Tamura, Tsuyoshi Fukushima, Shuhei Asada, Reina Takeda, Yuya Kunisaki, Tomofusa Fukuyama, Susumu Goyama, Kazuaki Yokoyama, Arinobu Tojo, Yoshio Katayama, Fumio Arai, Takashi Nagasawa, Takahiro Ochiya, Daichi Inoue, Impaired Osteoblastic Differentiation of MSCs Suppresses Normal Hematopoiesis in MDS, BLOOD, 10.1182/blood-2020-142284, 136, 2020.11, 0.
15. Hiroyasu Kidoya, Fumitaka Muramatsu, Teppei Shimamura, Weizhen Jia, Takashi Satoh, Yumiko Hayashi, Hisamichi Naito, Yuya Kunisaki, Fumio Arai, Masahide Seki, Yutaka Suzuki, Tsuyoshi Osawa, Shizuo Akira, Nobuyuki Takakura, Regnase-1-mediated post-transcriptional regulation is essential for hematopoietic stem and progenitor cell homeostasis, Nature communications, 10.1038/s41467-019-09028-w, 10, 1, 2019.12, The balance between self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) maintains hematopoietic homeostasis, failure of which can lead to hematopoietic disorder. HSPC fate is controlled by signals from the bone marrow niche resulting in alteration of the stem cell transcription network. Regnase-1, a member of the CCCH zinc finger protein family possessing RNAse activity, mediates post-transcriptional regulatory activity through degradation of target mRNAs. The precise function of Regnase-1 has been explored in inflammation-related cytokine expression but its function in hematopoiesis has not been elucidated. Here, we show that Regnase-1 regulates self-renewal of HSPCs through modulating the stability of Gata2 and Tal1 mRNA. In addition, we found that dysfunction of Regnase-1 leads to the rapid onset of abnormal hematopoiesis. Thus, our data reveal that Regnase-1-mediated post-transcriptional regulation is required for HSPC maintenance and suggest that it represents a leukemia tumor suppressor..
16. Hiroshi Kobayashi, Takayuki Morikawa, Ayumi Okinaga, Fumie Hamano, Tomomi Hashidate-Yoshida, Shintaro Watanuki, Daisuke Hishikawa, Hideo Shindou, Fumio Arai, Yasuaki Kabe, Makoto Suematsu, Takao Shimizu, Keiyo Takubo, Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cells Ex Vivo, Cell Reports, 10.1016/j.celrep.2019.06.008, 28, 1, 145-158.e9, 2019.07, Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our culture system provides a means to evaluate properties of steady-state HSCs and test effects of defined factors in vitro under near-physiological conditions. Maintaining quiescent HSCs under physiological conditions facilitates evaluation of the properties of steady-state HSCs. Kobayashi et al. report that low cytokine concentrations, hypoxia, and high fatty acid levels mimicking the bone marrow microenvironment enable maintenance of engraftable quiescent HSCs for 1 month in culture..
17. Kentaro Hosokawa, Fumio Arai, The role of telomere binding molecules for normal and abnormal hematopoiesis, International Journal of Hematology, 10.1007/s12185-018-2432-4, 107, 6, 646-655, 2018.06, In order to maintain the homeostasis of the hematopoietic system, hematopoietic stem cells (HSCs) need to be maintained while slowly dividing over their lifetime. However, repeated cell divisions lead to the gradual accumulation of DNA damage and ultimately impair HSC function. Since telomeres are particularly fragile when subjected to replication stress, cells have several defense machinery to protect telomeres. Moreover, HSCs must protect their genome against possible DNA damage, while maintaining telomere length. A group of proteins called the shelterin complex are deeply involved in this two-way role, and it is highly resistant to the replication stress to which HSCs are subjected. Most shelterin-deficient experimental models suffer acute cytotoxicity and severe phenotypes, as each shelterin component is essential for telomere protection. The Tin2 point mutant mice show a dyskeratosis congenita (DC) like phenotype, and the Tpp1 deletion impairs the hematopoietic system. POT1/Pot1a is highly expressed in HSCs and contributes to the maintenance of the HSC pool during in vitro culture. Here, we discuss the role of shelterin molecules in HSC regulation and review current understanding of how these are regulated in the maintenance of the HSC pool and the development of hematological disorders..
18. Fumio Arai, Guest editorial
Regulatory signaling in normal and abnormal hematopoiesis, International Journal of Hematology, 10.1007/s12185-018-2460-0, 1-3, 2018.05, Stem cells are characterized by their unique ability to both self-renew and differentiate along multiple cellular lineages. Self-renewal and differentiation must be tightly controlled to ensure an appropriate stem cell pool in tissue over the lifetime of an organism. Elucidating the mechanisms controlling stem cell fate and maintenance remains a key challenge in stem cell biology. Hematopoietic stem cells (HSCs) are responsible for the lifelong production of multiple blood cell lineages. To remain functional, these cells must interact with a particular microenvironment, known as the stem cell niche. HSC niches provide various factors, including cytokines, extracellular matrices, nutrients, hormones, and metabolites. These niche factors modulate cell-intrinsic molecular regulatory networks in HSCs. Niche signals also play crucial roles in the induction of HSCs from pluripotent stem cells or vascular endothelial cells. The Progress in Hematology review series in the current issue highlights some critical regulators of HSC maintenance and production..
19. Yuko Tadokoro, Takayuki Hoshii, Satoshi Yamazaki, Koji Eto, Hideo Ema, Masahiko Kobayashi, Masaya Ueno, Kumiko Ohta, Yuriko Arai, Eiji Hara, Kenichi Harada, Masanobu Oshima, Hiroko Oshima, Fumio Arai, Akihiko Yoshimura, Hiromitsu Nakauchi, Atsushi Hirao, Spred1 Safeguards Hematopoietic Homeostasis against Diet-Induced Systemic Stress, Cell Stem Cell, 10.1016/j.stem.2018.04.002, 22, 5, 713-725.e8, 2018.05, Stem cell self-renewal is critical for tissue homeostasis, and its dysregulation can lead to organ failure or tumorigenesis. While obesity can induce varied abnormalities in bone marrow components, it is unclear how diet might affect hematopoietic stem cell (HSC) self-renewal. Here, we show that Spred1, a negative regulator of RAS-MAPK signaling, safeguards HSC homeostasis in animals fed a high-fat diet (HFD). Under steady-state conditions, Spred1 negatively regulates HSC self-renewal and fitness, in part through Rho kinase activity. Spred1 deficiency mitigates HSC failure induced by infection mimetics and prolongs HSC lifespan, but it does not initiate leukemogenesis due to compensatory upregulation of Spred2. In contrast, HFD induces ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in functional HSC failure, severe anemia, and myeloproliferative neoplasm-like disease. HFD-induced hematopoietic abnormalities are mediated partly through alterations to the gut microbiota. Together, these findings reveal that diet-induced stress disrupts fine-tuning of Spred1-mediated signals to govern HSC homeostasis. Tadokoro et al. show that Spred1 negatively regulates HSC self-renewal in a manner supported by ROCK activity and that Spred1 safeguards HSC homeostasis under high-fat diet (HFD) conditions by regulating HSC self-renewal. The gut microbiota dysbiosis induced by HFD disrupts the fine-tuning of Spred1-mediated signals that govern HSC homeostasis..
20. takuji yamauchi, Takeshi Masuda, Matthew C. Canver, Michael Seiler, Yuichiro Semba, Mohammad Shboul, Mohammed Al-Raqad, Manami Maeda, Vivien A.C. Schoonenberg, Mitchel A. Cole, Claudio Macias-Trevino, Yuichi Ishikawa, Qiuming Yao, Michitaka Nakano, Fumio Arai, Stuart H. Orkin, Bruno Reversade, Silvia Buonamici, Luca Pinello, Koichi Akashi, Daniel E. Bauer, Takahiro Maeda, Genome-wide CRISPR-Cas9 Screen Identifies Leukemia-Specific Dependence on a Pre-mRNA Metabolic Pathway Regulated by DCPS, Cancer Cell, 10.1016/j.ccell.2018.01.012, 33, 3, 386-400.e5, 2018.03, To identify novel targets for acute myeloid leukemia (AML) therapy, we performed genome-wide CRISPR-Cas9 screening using AML cell lines, followed by a second screen in vivo. Here, we show that the mRNA decapping enzyme scavenger (DCPS) gene is essential for AML cell survival. The DCPS enzyme interacted with components of pre-mRNA metabolic pathways, including spliceosomes, as revealed by mass spectrometry. RG3039, a DCPS inhibitor originally developed to treat spinal muscular atrophy, exhibited anti-leukemic activity via inducing pre-mRNA mis-splicing. Humans harboring germline biallelic DCPS loss-of-function mutations do not exhibit aberrant hematologic phenotypes, indicating that DCPS is dispensable for human hematopoiesis. Our findings shed light on a pre-mRNA metabolic pathway and identify DCPS as a target for AML therapy. Yamauchi et al. perform in vitro and in vivo CRISPR-Cas9 genetic screening of p53 WT AML to identify potential therapeutic targets. They find that AML relies on the DCPS decapping enzyme, and a DCPS inhibitor shows anti-leukemia activity in tumor models without impacting normal hematopoiesis..
21. Mohamed A.E. Ali, Kyoko Fuse, Yuko Tadokoro, Takayuki Hoshii, Masaya Ueno, Masahiko Kobayashi, Naho Nomura, Ha Thi Vu, Hui Peng, Ahmed M. Hegazy, Masayoshi Masuko, Hirohito Sone, Fumio Arai, Atsushi Tajima, Atsushi Hirao, Functional dissection of hematopoietic stem cell populations with a stemness-monitoring system based on NS-GFP transgene expression, Scientific Reports, 10.1038/s41598-017-11909-3, 7, 1, 2017.12, Hematopoietic stem cells (HSCs) in a steady state can be efficiently purified by selecting for a combination of several cell surface markers; however, such markers do not consistently reflect HSC activity. In this study, we successfully enriched HSCs with a unique stemness-monitoring system using a transgenic mouse in which green florescence protein (GFP) is driven by the promoter/enhancer region of the nucleostemin (NS) gene. We found that the phenotypically defined long-term (LT)-HSC population exhibited the highest level of NS-GFP intensity, whereas NS-GFP intensity was strongly downregulated during differentiation in vitro and in vivo. Within the LT-HSC population, NS-GFPhigh cells exhibited significantly higher repopulating capacity than NS-GFPlow cells. Gene expression analysis revealed that nine genes, including Vwf and Cdkn1c (p57), are highly expressed in NS-GFPhigh cells and may represent a signature of HSCs, i.e., a stemness signature. When LT-HSCs suffered from remarkable stress, such as transplantation or irradiation, NS-GFP intensity was downregulated. Finally, we found that high levels of NS-GFP identified HSC-like cells even among CD34+ cells, which have been considered progenitor cells without long-term reconstitution ability. Thus, high NS-GFP expression represents stem cell characteristics in hematopoietic cells, making this system useful for identifying previously uncharacterized HSCs..
22. Kentaro Hosokawa, Ben D. MacArthur, Yoshiko Matsumoto Ikushima, Hirofumi Toyama, Yoshikazu Masuhiro, Shigemasa Hanazawa, Toshio Suda, Fumio Arai, The telomere binding protein Pot1 maintains haematopoietic stem cell activity with age., Nature Communications, 10.1038/s41467-017-00935-4, 8, 1, 2017.10, Repeated cell divisions and aging impair stem cell function. However, the mechanisms by　which this occurs are not fully understood. Here we show that protection of telomeres 1A　(Pot1a), a component of the Shelterin complex that protects telomeres, improves haematopoietic　stem cell (HSC) activity during aging. Pot1a is highly expressed in young HSCs, but
declines with age. In mouse HSCs, Pot1a knockdown increases DNA damage response (DDR)　and inhibits self-renewal. Conversely, Pot1a overexpression or treatment with POT1a protein　prevents DDR, maintained self-renewal activity and rejuvenated aged HSCs upon ex vivo　culture. Moreover, treatment of HSCs with exogenous Pot1a inhibits the production of reactive oxygen species, suggesting a non-telomeric role for Pot1a in HSC maintenance.　Consistent with these results, treatment with exogenous human POT1 protein maintains　human HSC activity in culture. Collectively, these results show that Pot1a/POT1 sustains HSC　activity and can be used to expand HSC numbers ex vivo..
23. Patrick S. Stumpf, Rosanna C.G. Smith, Michael Lenz, Andreas Schuppert, Franz Josef Müller, Ann Babtie, Thalia E. Chan, Michael P.H. Stumpf, Colin P. Please, Sam D. Howison, Fumio Arai, Ben D. MacArthur, Stem Cell Differentiation as a Non-Markov Stochastic Process, Cell Systems, 10.1016/j.cels.2017.08.009, 5, 3, 268-282.e7, 2017.09, Pluripotent stem cells can self-renew in culture and differentiate along all somatic lineages in vivo. While much is known about the molecular basis of pluripotency, the mechanisms of differentiation remain unclear. Here, we profile individual mouse embryonic stem cells as they progress along the neuronal lineage. We observe that cells pass from the pluripotent state to the neuronal state via an intermediate epiblast-like state. However, analysis of the rate at which cells enter and exit these observed cell states using a hidden Markov model indicates the presence of a chain of unobserved molecular states that each cell transits through stochastically in sequence. This chain of hidden states allows individual cells to record their position on the differentiation trajectory, thereby encoding a simple form of cellular memory. We suggest a statistical mechanics interpretation of these results that distinguishes between functionally distinct cellular “macrostates” and functionally similar molecular “microstates” and propose a model of stem cell differentiation as a non-Markov stochastic process. We profile individual mouse embryonic stem cells as they progress along the neuronal lineage. Analysis of observed cell dynamics using a hidden Markov model reveals the presence of a chain of unobserved molecular states that each cell transits through stochastically in sequence. We suggest a statistical mechanics interpretation of these results and propose a model of stem cell differentiation as a non-Markov stochastic process..
24. Kyoko Ito, Raphaël Turcotte, Jinhua Cui, Samuel E. Zimmerman, Sandra Pinho, Toshihide Mizoguchi, Fumio Arai, Judith M. Runnels, Clemens Alt, Julie Teruya-Feldstein, Jessica C. Mar, Rajat Singh, Toshio Suda, Charles P. Lin, Paul S. Frenette, Keisuke Ito, Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance, Science, 10.1126/science.aaf5530, 354, 6316, 1156-1160, 2016.12, A single hematopoietic stem cell (HSC) is capable of reconstituting hematopoiesis and maintaining homeostasis by balancing self-renewal and cell differentiation. The mechanisms of HSC division balance, however, are not yet defined. Here we demonstrate, by characterizing at the single-cell level a purified and minimally heterogeneous murine Tie2+ HSC population, that these top hierarchical HSCs preferentially undergo symmetric divisions. The induction of mitophagy, a quality control process in mitochondria, plays an essential role in self-renewing expansion of Tie2+ HSCs. Activation of the PPAR (peroxisome proliferator-activated receptor)-fatty acid oxidation pathway promotes expansion of Tie2+ HSCs through enhanced Parkin recruitment in mitochondria. These metabolic pathways are conserved in human TIE2+ HSCs. Our data thus identify mitophagy as a key mechanism of HSC expansion and suggest potential methods of cell-fate manipulation through metabolic pathways..
25. Self-renewal and differentiation of hematopoietic stem cells.
26. Shuhei Koide, Motohiko Oshima, Keiyo Takubo, Satoshi Yamazaki, Eriko Nitta, Atsunori Saraya, Kazumasa Aoyama, Yuko Kato, Satoru Miyagi, Yaeko Nakajima-Takagi, Tetsuhiro Chiba, Hirotaka Matsui, Fumio Arai, Yutaka Suzuki, Hiroshi Kimura, Hiromitsu Nakauchi, Toshio Suda, Yoichi Shinkai, Atsushi Iwama, Setdb1 maintains hematopoietic stem and progenitor cells by restricting the ectopic activation of nonhematopoietic genes, Blood, 10.1182/blood-2016-01-694810, 128, 5, 638-649, 2016.08, Setdb1, also known as Eset, is a methyltransferase that catalyzes trimethylation of H3K9 (H3K9me3) and plays an essential role in the silencing of endogenous retroviral elements (ERVs) in the developing embryo and embryonic stem cells (ESCs). Its role in somatic stem cells, however, remains unclear because of the early death of Setdb1-deficient embryos. We demonstrate here that Setdb1 is the first H3K9 methyltransferase shown to be essential for the maintenance of hematopoietic stem and progenitor cells (HSPCs) in mice. The deletion of Setdb1 caused the rapid depletion of hematopoietic stem and progenitor cells (HSPCs), as well as leukemic stem cells. In contrast to ESCs, ERVs were largely repressed in Setdb1-deficient HSPCs. A list of nonhematopoietic genes was instead ectopically activated in HSPCs after reductions in H3K9me3 levels, including key gluconeogenic enzyme genes fructose-1,6-bisphosphatase 1 (Fbp1) and Fbp2. The ectopic activation of gluconeogenic enzymes antagonized glycolysis and impaired ATP production, resulting in a compromised repopulating capacity of HSPCs. Our results demonstrate that Setdb1 maintains HSPCs by restricting the ectopic activation of nonhematopoietic genes detrimental to their function and uncover that the gluconeogenic pathway is one of the critical targets of Setdb1 in HSPCs..
27. Hiroshi Sakamoto, Naoki Takeda, Fumio Arai, Kentaro Hosokawa, Paloma Garcia, Toshio Suda, Jon Frampton, Minetaro Ogawa, Determining c-Myb protein levels can isolate functional hematopoietic stem cell subtypes, Stem Cells, 10.1002/stem.1855, 33, 2, 479-490, 2015.01, The transcription factor c-Myb was originally identified as a transforming oncoprotein encoded by two avian leukemia viruses. Subsequently, through the generation of mouse models that affect its expression, c-Myb has been shown to be a key regulator of hematopoiesis, including having critical roles in hematopoietic stem cells (HSCs). The precise function of c-Myb in HSCs although remains unclear. We have generated a novel c-myb allele in mice that allows direct observation of c-Myb protein levels in single cells. Using this reporter line we demonstrate that subtypes of HSCs can be isolated based upon their respective c-Myb protein expression levels. HSCs expressing low levels of c-Myb protein (c-MyblowHSC) appear to represent the most immature, dormant HSCs and they are a predominant component of HSCs that retain bromodeoxyuridine labeling. Hematopoietic stress, induced by 5-fluorouracil ablation, revealed that in this circumstance c-Myb-expressing cells become critical for multilineage repopulation. The discrimination of HSC subpopulations based on c-Myb protein levels is not reflected in the levels of c-myb mRNA, there being no more than a 1.3-fold difference comparing c-Myblow and c-MybhighHSCs. This illustrates how essential it is to include protein studies when aiming to understand the regulatory networks that control stem cell behavior..
28. Masayuki Yamashita, Eriko Nitta, Go Nagamatsu, Yoshiko Matsumoto Ikushima, Kentaro Hosokawa, Fumio Arai, Toshio Suda, Nucleostemin is indispensable for the maintenance and genetic stability of hematopoietic stem cells, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2013.10.032, 441, 1, 196-201, 2013.11, Nucleostemin is a nucleolar protein known to play a variety of roles in cell-cycle progression, apoptosis inhibition, and DNA damage protection in embryonic stem cells and tissue stem cells. However, the role of nucleostemin in hematopoietic stem cells (HSCs) is yet to be determined. Here, we identified an indispensable role of nucleostemin in mouse HSCs. Depletion of nucleostemin using short hairpin RNA strikingly impaired the self-renewal activity of HSCs both in vitro and in vivo. Consistently, nucleostemin depletion triggered apoptosis rather than cell-cycle arrest in HSCs. Furthermore, DNA damage accumulated during cultivation upon depletion of nucleostemin. The impaired self-renewal activity of HSCs induced by nucleostemin depletion was partially rescued by p53 deficiency but not by p16Ink4a or p19Arf deficiency. Taken together, our study demonstrates that nucleostemin protects HSCs from DNA damage accumulation and is required for the maintenance of HSCs..
29. Yoshiko Matsumoto Ikushima, Fumio Arai, Kentaro Hosokawa, Hirofumi Toyama, Keiyo Takubo, Tomoyuki Furuyashiki, Shuh Narumiya, Toshio Suda, Prostaglandin E2 regulates murine hematopoietic stem/progenitor cells directly via EP4 receptor and indirectly through mesenchymal progenitor cells, Blood, 10.1182/blood-2012-06-437889, 121, 11, 1995-2007, 2013.03, Key Points
PGE2 signaling positively regulates hematopoietic stem cells both directly and via activation of a nonhematopoietic cell population. EP4 is a major receptor for the PGE2-mediated regulation of hematopoietic stem and progenitor cells..
30. Yumiko Matsubara, Yukako Ono, Hidenori Suzuki, Fumio Arai, Toshio Suda, Mitsuru Murata, Yasuo Ikeda, OP9 Bone Marrow Stroma Cells Differentiate into Megakaryocytes and Platelets, PLoS One, 10.1371/journal.pone.0058123, 8, 3, 2013.03, Platelets are essential for hemostatic plug formation and thrombosis. The mechanisms of megakaryocyte (MK) differentiation and subsequent platelet production from stem cells remain only partially understood. The manufacture of megakaryocytes (MKs) and platelets from cell sources including hematopoietic stem cells and pluripotent stem cells have been highlighted for studying the platelet production mechanisms as well as for the development of new strategies for platelet transfusion. The mouse bone marrow stroma cell line OP9 has been widely used as feeder cells for the differentiation of stem cells into MK lineages. OP9 cells are reported to be pre-adipocytes. We previously reported that 3T3-L1 pre-adipocytes differentiated into MKs and platelets. In the present study, we examined whether OP9 cells differentiate into MKs and platelets using MK lineage induction (MKLI) medium previously established to generate MKs and platelets from hematopoietic stem cells, embryonic stem cells, and pre-adipocytes. OP9 cells cultured in MKLI medium had megakaryocytic features, i.e., positivity for surface markers CD41 and CD42b, polyploidy, and distinct morphology. The OP9-derived platelets had functional characteristics, providing the first evidence for the differentiation of OP9 cells into MKs and platelets. We then analyzed gene expressions of critical factors that regulate megakaryopoiesis and thrombopoiesis. The gene expressions of p45NF-E2, FOG, Fli1, GATA2, RUNX1, thrombopoietin, and c-mpl were observed during the MK differentiation. Among the observed transcription factors of MK lineages, p45NF-E2 expression was increased during differentiation. We further studied MK and platelet generation using p45NF-E2-overexpressing OP9 cells. OP9 cells transfected with p45NF-E2 had enhanced production of MKs and platelets. Our findings revealed that OP9 cells differentiated into MKs and platelets in vitro. OP9 cells have critical factors for megakaryopoiesis and thrombopoiesis, which might be involved in a mechanism of this differentiation. p45NF-E2 might also play important roles in the differentiation of OP9 cells into MK lineages cells..
31. Yoshiko Matsumoto Ikushima, Fumio Arai, Yuka Nakamura, Kentaro Hosokawa, Yoshiaki Kubota, Masanori Hirashima, Hirofumi Toyama, Toshio Suda, Enhanced Angpt1/Tie2 signaling affects the differentiation and long-term repopulation ability of hematopoietic stem cells, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2012.11.002, 430, 1, 20-25, 2013.01, Angiopoietin-1 (Angpt1) signaling via the Tie2 receptor regulates vascular and hematopoietic systems. To investigate the role of Angpt1-Tie2 signaling in hematopoiesis, we prepared conditionally inducible transgenic (Tg) mice expressing a genetically engineered Angpt1, cartridge oligomeric matrix protein (COMP)-Angpt1. The effects of COMP-Angpt1 overexpression in osteoblasts on hematopoiesis were then investigated by crossing COMP-Angpt1 Tg mice with Col1a1-Cre Tg mice. Interestingly, peripheral blood analyses showed that 4week (wk)-old (but not 8 wk-old) Col1a1-Cre+/COMP-Angpt1+ mice had a lower percentage of circulating B cells and a higher percentage of myeloid cells than Col1a1-Cre-/COMP-Angpt1+ (control) mice. Although there were no significant differences in the immunophenotypic hematopoietic stem and progenitor cell (HSPC) populations between Col1a1-Cre+/COMP-Angpt1+ and control mice, lineage-Sca-1+c-Kit+ (LSK) cells isolated from 8 wk-old Col1a1-Cre+/COMP-Angpt1+ mice showed better long-term bone marrow reconstitution ability. These data indicate that Angpt1-Tie2 signaling affects the differentiation capacity of hematopoietic lineages during development and increases the stem cell activity of HSCs..
32. Hirofumi Toyama, Fumio Arai, Kentaro Hosokawa, Yoshiko Matsumoto Ikushima, Toshio Suda, N-cadherin+ HSCs in fetal liver exhibit higher long-term bone marrow reconstitution activity than N-cadherin- HSCs, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2012.10.058, 428, 3, 354-359, 2012.11, Adult hematopoietic stem cells (HSCs) are maintained in a microenvironment known as the stem cell niche. The regulation of HSCs in fetal liver (FL) and their niche, however, remains to be elucidated. In this study, we investigated the role of N-cadherin (N-cad) in the maintenance of HSCs during FL hematopoiesis. By using anti-N-cad antibodies (Abs) produced by our laboratory, we detected high N-cad expression in embryonic day 12.5 (E12.5) mouse FL HSCs, but not in E15.5 and E18.5 FL. Immunofluorescence staining revealed that N-cad+c-Kit+ and N-cad+ endothelial protein C receptor (EPCR)+ HSCs co-localized with Lyve-1+ sinusoidal endothelial cells (ECs) in E12.5 FL and that some of these cells also expressed N-cad. However, N-cad+ HSCs were also observed to detach from the perisinusoidal niche at E15.5 and E18.5, concomitant with a down-regulation of N-cad and an up-regulation of E-cadherin (E-cad) in hepatic cells. Moreover, EPCR+ long-term (LT)-HSCs were enriched in the N-cad+Lin-Sca-1+c-Kit+ (LSK) fraction in E12.5 FL, but not in E15.5 or E18.5 FL. In a long-term reconstitution (LTR) activity assay, higher engraftment associated with N-cad+ LSK cells versus N-cad- LSK cells in E12.5 FL when transplanted into lethally irradiated recipient mice. However, the higher engraftment of N-cad+ LSK cells decreased subsequently in E15.5 and E18.5 FL. It is possible that N-cad expression conferred higher LTR activity to HSCs by facilitating interactions with the perisinusoidal niche, especially at E12.5. The down-regulation of N-cad during FL hematopoiesis may help us better understand the regulation and mobility of HSCs before migration into BM. © 2012 Elsevier Inc..
33. Keisuke Ito, Arkaitz Carracedo, Dror Weiss, Fumio Arai, Ugo Ala, David E. Avigan, Zachary T. Schafer, Ronald M. Evans, Toshio Suda, Chih Hao Lee, Pier Paolo Pandolfi, A PML-PPAR-δ pathway for fatty acid oxidation regulates hematopoietic stem cell maintenance, Nature Medicine, 10.1038/nm.2882, 18, 9, 1350-1358, 2012.09, Stem-cell function is an exquisitely regulated process. Thus far, the contribution of metabolic cues to stem-cell function has not been well understood. Here we identify a previously unknown promyelocytic leukemia (PML)-peroxisome proliferator-activated receptor δ (PPAR-δ)-fatty- acid oxidation (FAO) pathway for the maintenance of hematopoietic stem cells (HSCs). We have found that loss of PPAR-δ or inhibition of mitochondrial FAO induces loss of HSC maintenance, whereas treatment with PPAR-δ agonists improved HSC maintenance. PML exerts its essential role in HSC maintenance through regulation of PPAR signaling and FAO. Mechanistically, the PML-PPAR-δ-FAO pathway controls the asymmetric division of HSCs. Deletion of Ppard or Pml as well as inhibition of FAO results in the symmetric commitment of HSC daughter cells, whereas PPAR-δ activation increased asymmetric cell division. Thus, our findings identify a metabolic switch for the control of HSC cell fate with potential therapeutic implications..
34. Ryohichi Sugimura, Xi C. He, Aparna Venkatraman, Fumio Arai, Andrew Box, Craig Semerad, Jeffrey S. Haug, Lai Peng, Xiao Bo Zhong, Toshio Suda, Linheng Li, Noncanonical Wnt signaling maintains hematopoietic stem cells in the niche, Cell, 10.1016/j.cell.2012.05.041, 150, 2, 351-365, 2012.07, Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin+ osteoblasts (N-cad +OBs that enrich osteoprogenitors) in the niche. We further found that N-cad+OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca2+-NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche..
35. Fumio Arai, Kentaro Hosokawa, Yoshiko Matsumoto, Hirofumi Toyama, Toshio Suda, Gene expression profiling and regulatory networks in single cells, New Frontiers of Network Analysis in Systems Biology, 10.1007/978-94-007-4330-4_1, 1-13, 2012.02, Tissues and organs are composed of various kinds of cells including stem cells, progenitors, and terminally differentiated cells that have large variations of gene expression. Within a seemingly homogeneous cell population, gene expression levels may differ dramatically on a cell-to-cell level. However, differences or variations of individual cells are masked by the averaging effect of pooled samples in population analysis. Therefore, analyzing transcript levels for multiple genes across multiple individual cells could be key for understanding the unique characteristics of individual cells and for clarifying the complicated mechanisms controlling the function of individual cells. Recent advances in cDNA amplification techniques from single cell samples allow for analyzing gene expression patterns in individual cells. Amplified cDNAs are applicable to measurements of gene expression systems such as microarrays, DNA sequencing, and TaqMan assay-based high throughput nanofluidic real time PCR array analysis. Gene expression profiling at the single cell level allows us to identify specific sub-populations in heterogeneous cell populations in tissues and clarify the complex networks controlling the function of individual cells..
36. Shabnam Kharazi, Adam J. Mead, Anna Mansour, Anne Hultquist, Charlotta Böiers, Sidinh Luc, Natalija Buza-Vidas, Zhi Ma, Helen Ferry, Debbie Atkinson, Kristian Reckzeh, Kristina Masson, Jörg Cammenga, Lars Rönnstrand, Fumio Arai, Toshio Suda, Claus Nerlov, Ewa Sitnicka, Sten Eirik W. Jacobsen, Impact of gene dosage, loss of wild-type allele, and FLT3 ligand on Flt3-ITD-induced myeloproliferation, Blood, 10.1182/blood-2010-06-289207, 118, 13, 3613-3621, 2011.09, Acquisition of homozygous activating growth factor receptor mutations might accelerate cancer progression through a simple gene-dosage effect. Internal tandem duplications (ITDs) of FLT3 occur in approximately 25% cases of acute myeloid leukemia and induce ligand-independent constitutive signaling. Homozygous FLT3-ITDs confer an adverse prognosis and are frequently detected at relapse. Using a mouse knockin model of Flt3 - internal tandem duplication (Flt3-ITD) - induced myeloproliferation, we herein demonstrate that the enhanced myeloid phenotype and expansion of granulocyte-monocyte and primitive Lin -Sca1 +c-Kit + progenitors in Flt3-ITD homozygous mice can in part be mediated through the loss of the second wild-type allele. Further, whereas autocrine FLT3 ligand production has been implicated in FLT3-ITD myeloid malignancies and resistance to FLT3 inhibitors, we demonstrate here that the mouse Flt3 ITD/ITD myeloid phenotype is FLT3 ligand-independent..
37. Eriko Nitta, Masayuki Yamashita, Kentaro Hosokawa, Ming Ji Xian, Keiyo Takubo, Fumio Arai, Shinichiro Nakada, Toshio Suda, Telomerase reverse transcriptase protectsATM-deficient hematopoietic stem cells from ROS-induced apoptosis through a telomere-independent mechanism, Blood, 10.1182/blood-2010-08-297390, 117, 16, 4169-4180, 2011.04, Telomerase reverse transcriptase (TERT) contributes to the prevention of aging by a largely unknown mechanism that is unrelated to telomere lengthening. The current study used ataxia-telangiectasia mutated (ATM) and TERT doubly deficient mice to evaluate the contributions of 2 aging-regulating molecules, TERT and ATM, to the aging process. ATM and TERT doubly deficient mice demonstrated increased progression of aging and had shorter lifespans than ATM-null mice, while TERT alone was insufficient to affect lifespan. ATM-TERT doubly null mice show in vivo senescence, especially in hematopoietic tissues, that was dependent on p16 INK4a and p19 ARF, but not on p21. As their HSCs show decreased stem cell activities, accelerated aging seen in these mice has been attributed to impaired stem cell function. TERT-deficient HSCs are characterized by reactive oxygen species (ROS) fragility, which has been suggested to cause stem cell impairment during aging, and apoptotic HSCs are markedly increased in these mice. p38MAPK activation was indicated to be partially involved in ROS-induced apoptosis in TERT-null HSCs, and BCL-2 is suggested to provide a part of the protective mechanisms of HSCs by TERT. The current study demonstrates that TERT mitigates aging by protecting HSCs under stressful conditions through telomere length-independent mechanisms..
38. Haruko Shima, Keiyo Takubo, Naoko Tago, Hiroko Iwasaki, Fumio Arai, Takao Takahashi, Toshio Suda, Acquisition of G0 state by CD34-positive cord blood cells after bone marrow transplantation, Experimental Hematology, 10.1016/j.exphem.2010.08.004, 38, 12, 1231-1240, 2010.12, Objective: Hematopoietic stem cells are kept in a quiescent state in the hypoxic area of the bone marrow, which is essential for hematopoietic stem cell maintenance. However, when and how hematopoietic stem cells acquire their hypoxic state and maintain quiescence has not been fully elucidated. The aim of this study was to understand this process in human hematopoietic stem cells after bone marrow transplantation. Materials and Methods: Human CD34-positive cord blood cells were transplanted into nonobese diabetic/severe combined immunodeficient interleukin-2 receptor γ chain knockout mice. Cell cycle and hypoxia assay analyses were performed, to identify changes in the characteristics of human hematopoietic stem cells following transplantation. Quantitative real-time reverse transcription polymerase chain reaction analysis was used to analyze the transcriptional changes accompanying this transition. Results: Engrafted primitive lineage-negative CD34-positive CD38-negative cells acquired hypoxic state and quiescence in the recipient bone marrow between 4 and 8 weeks, and between 8 and 12 weeks after transplantation, respectively. During 4 and 8 weeks after transplantation, changes in the transcription levels of G0 regulatory factors, such as CCNC and RBL1, and stem cell regulators, such as Flt3, were also seen, which may be related to the characteristic changes in the cell cycle or oxygenation state. Conclusions: Behavioral changes of hematopoietic stem cells in their cell cycle and oxygenation state during and after bone marrow engraftment may provide insights into hematopoietic stem cell regulation, mediating the improvement of clinical hematopoietic stem cell transplantation protocols and the eradication of leukemia stem cells..
39. Hideaki Nakajima, Miyuki Ito, David S. Smookler, Fumi Shibata, Yumi Fukuchi, Yoshihiro Morikawa, Yuichi Ikeda, Fumio Arai, Toshio Suda, Rama Khokha, Toshio Kitamura, TIMP-3 recruits quiescent hematopoietic stem cells into active cell cycle and expands multipotent progenitor pool, Blood, 10.1182/blood-2010-01-266528, 116, 22, 4474-4482, 2010.11, Regulating transition of hematopoietic stem cells (HSCs) between quiescent and cycling states is critical for maintaining homeostasis of blood cell production. The cycling states of HSCs are regulated by the extracellular factors such as cytokines and extracellular matrix; however, the molecular circuitry for such regulation remains elusive. Here we show that tissue inhibitor of metalloproteinase-3 (TIMP-3), an endogenous regulator of metalloproteinases, stimulates HSC proliferation by recruiting quiescent HSCs into the cell cycle. Myelosuppression induced TIMP-3 in the bone marrow before hematopoietic recovery. Interestingly, TIMP-3 enhanced proliferation of HSCs and promoted expansion of multipotent progenitors, which was achieved by stimulating cell-cycle entry of quiescent HSCs without compensating their long-term repopulating activity. Surprisingly, this effect did not require metalloproteinase inhibitory activity of TIMP-3 and was possibly mediated through a direct inhibition of angiopoietin-1 signaling, a critical mediator for HSC quiescence. Furthermore, bone marrow recovery from myelosuppression was accelerated by over-expression of TIMP-3, and in turn, impaired in TIMP-3 - deficient animals. These results suggest that TIMP-3 may act as a molecular cue in response to myelosuppression for recruiting dormant HSCs into active cell cycle and may be clinically useful for facilitating hematopoietic recovery after chemotherapy or ex vivo expansion of HSCs..
40. Yuka Nakamura, Fumio Arai, Hiroko Iwasaki, Kentaro Hosokawa, Isao Kobayashi, Yumiko Gomei, Yoshiko Matsumoto, Hiroki Yoshihara, Toshio Suda, Isolation and characterization of endosteal niche cell populations that regulate hematopoietic stem cells, Blood, 10.1182/blood-2009-08-239194, 116, 9, 1422-1432, 2010.09, The endosteal niche is critical for the maintenance of hematopoietic stem cells (HSCs). However, it consists of a heterogeneous population in terms of differentiation stage and function. In this study, we characterized endosteal cell populations and examined their ability to maintain HSCs. Bone marrow endosteal cells were subdivided into immature mesenchymal cell-enriched ALCAM-Sca-1+ cells, osteoblast-enriched ALCAM +Sca-1-, and ALCAM-Sca-1- cells. We found that all 3 fractions maintained long-term reconstitution (LTR) activity of HSCs in an in vitro culture. In particular, ALCAM+Sca-1- cells significantly enhanced the LTR activity of HSCs by the up-regulation of homing-and cell adhesion-related genes in HSCs. Microarray analysis showed that ALCAM-Sca-1+ fraction highly expressed cytokine-related genes, whereas the ALCAM+Sca-1- fraction expressed multiple cell adhesion molecules, such as cadherins, at a greater level than the other fractions, indicating that the interaction between HSCs and osteoblasts via cell adhesion molecules enhanced the LTR activity of HSCs. Furthermore, we found an osteoblastic markerlow/- subpopulation in ALCAM +Sca-1- fraction that expressed cytokines, such as Angpt1 and Thpo, and stem cell marker genes. Altogether, these data suggest that multiple subsets of osteoblasts and mesenchymal progenitor cells constitute the endosteal niche and regulate HSCs in adult bone marrow..
41. Hiroko Iwasaki, Fumio Arai, Yoshiaki Kubota, Maria Dahl, Toshio Suda, Endothelial protein C receptor-expressing hematopoietic stem cells reside in the perisinusoidal niche in fetal liver, Blood, 10.1182/blood-2009-08-240903, 116, 4, 544-553, 2010.07, Hematopoietic stem cells (HSCs) are maintained in specialized niches in adult bone marrow. However, niche and HSC maintenance mechanism in fetal liver (FL) still remains unclear. Here, we investigated the niche and the molecular mechanism of HSC maintenance in mouse FL using HSCs expressing endothelial protein C receptor (EPCR). The antiapoptotic effect of activated protein C (APC) on EPCR+ HSCs and the expression of protease-activated receptor 1 (Par-1) mRNA in these cells suggested the involvement of the cytoprotective APC/ EPCR/Par-1 pathway in HSC maintenance. Immunohistochemistry revealed that EPCR+ cells were localized adjacent to, or integrated in, the Lyve-1+ sinusoidal network, whereAPC and extracellular matrix (ECM) are abundant, suggesting that HSCs in FL were maintained in the APC- and ECM-rich perisinusoidal niche. EPCR+ HSCs were in a relatively slow cycling state, consistent with their high expression levels of p57 and p18. Furthermore, the long-term reconstitution activity of EPCR+ HSCs decreased significantly after short culture but not when cocultured with feeder layer of FL-derived Lyve-1+ cells, which suggests that the maintenance of the self-renewal activity of FL HSCs largely depended on the interaction with the perisinusoidal niche. In conclusion, EPCR+ HSCs resided in the perisinusoidal niche in mouse FL..
42. Kentaro Hosokawa, Fumio Arai, Hiroki Yoshihara, Hiroko Iwasaki, Yuka Nakamura, Yumiko Gomei, Toshio Suda, Knockdown of N-cadherin suppresses the long-term engraftment of hematopoietic stem cells, Blood, 10.1182/blood-2009-05-224857, 116, 4, 554-563, 2010.07, During postnatal life, the bone marrow (BM) supports both self-renewal and differentiation of hematopoietic stem cells (HSCs) in specialized microenvironments termed stem cell niches. Cell-cell and cell-extracellular matrix interactions between HSCs and their niches are critical for the maintenance of HSC properties. Here, we analyzed the function of Ncadherin in the regulation of the proliferation and long-term repopulation activity of hematopoietic stem/progenitor cells (HSPCs) by the transduction of N-cadherin shRNA. Inhibition of N-cadherin expression accelerated cell division in vitro and reduced the lodgment of donor HSPCs to the endosteal surface, resulting in a significant reduction in long-term engraftment. Cotransduction of N-cadherin shRNA and a mutant N-cadherin that introduced the silent mutations to shRNA target sequences rescued the accelerated cell division and reconstitution phenotypes. In addition, the requirement of N-cadherin for HSPC engraftment appears to be niche specific, as shN-cad-transduced lineage-Sca- 1+c-Kit+ cells successfully engrafted in spleen, which lacks an osteoblastic niche. These findings suggest that N-cad-mediated cell adhesion is functionally required for the establishment of hematopoiesis in the BM niche after BM transplantation..
43. Kentaro Hosokawa, Fumio Arai, Hiroki Yoshihara, Hiroko Iwasaki, Mark Hembree, Tong Yin, Yuka Nakamura, Yumiko Gomei, Keiyo Takubo, Haruko Shiama, Sahoko Matsuoka, Linheng Li, Toshio Suda, Cadherin-Based Adhesion Is a Potential Target for Niche Manipulation to Protect Hematopoietic Stem Cells in Adult Bone Marrow, Cell stem cell, 10.1016/j.stem.2009.04.013, 6, 3, 194-198, 2010.03.
44. Keisuke Ito, Atsushi Hirao, Fumio Arai, Keiyo Takubo, Sahoko Matsuoka, Kana Miyamoto, Masako Ohmura, Kazuhito Naka, Kentaro Hosokawa, Yasuo Ikeda, Toshio Suda, Erratum
Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells (Nature Medicine (2006)12 (446-451)), Nature medicine, 10.1038/nm0110-129a, 16, 1, 2010.01.
45. Yumiko Gomei, Yuka Nakamura, Hiroki Yoshihara, Kentaro Hosokawa, Hiroko Iwasaki, Toshio Suda, Fumio Arai, Functional differences between two Tie2 ligands, angiopoietin-1 and -2, in regulation of adult bone marrow hematopoietic stem cells, Experimental Hematology, 10.1016/j.exphem.2009.11.007, 38, 2, 2010.01, Objective: Angiopoietin-1 (Ang-1) plays a critical role in the maintenance of hematopoietic stem cells (HSCs) in the bone marrow (BM) through its binding to the Tie2 receptor. Ang-2, another Tie2 ligand, is known to be an antagonist of Tie2/Ang-1 signaling in angiogenesis; however, its function in regulation of HSCs remains unclear. Here, we investigated the functional differences between Ang-1 and Ang-2 in the maintenance of HSCs. Materials and Methods: We treated mouse BM lineage
-
Sca-1
+
c-Kit
+
side population
+
cells with Ang-1 and/or Ang-2, and evaluated angiopoietin function by gene expression analysis, immunocytochemical staining of phosphorylated Akt, a colony-formation assay, and a long-term BM reconstitution assay. Results: Gene expression analysis and BM transplantation assay revealed that Ang-1 upregulated expression of p57, p18, Itgb1, Alcam, Tie2, Hoxb4, and Bmi1 genes in HSCs, while Ang-2 antagonized the effects of Ang-1. Ang-1 enhanced the phosphorylation of Akt, while Ang-2 again reduced the effect of Ang-1. The colony assay demonstrated that neither Ang-1, nor Ang-2 influenced the colony formation of HSCs. BM transplantation assay, following in vitro cultivation of HSCs with angiopoietins, showed that Ang-1 maintained long-term repopulating activity of HSCs, while the addition of Ang-2 interfered drastically with the effects of Ang-1. Conclusion: Gene expression analysis and BM transplantation assay demonstrated that Ang-1 maintained HSC activity in an in vitro culture. In contrast, Ang-2 reversed the effects of Ang-1/Tie2 signaling in the regulation of long-term HSCs. Our data suggest that Ang-1 is a dominant ligand for the Tie2 receptor in long HSCs in BM..
46. Tai Hato, Yoshishige Kimura, Tohru Morisada, Gou Young Koh, Keishi Miyata, Mitsuhisa Tabata, Tsuyoshi Kadomatsu, Motoyoshi Endo, Takashi Urano, Fumio Arai, Kimi Araki, Toshio Suda, Koichi Kobayashi, Yuichi Oike, Angiopoietins contribute to lung development by regulating pulmonary vascular network formation, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2009.02.030, 381, 2, 218-223, 2009.04, Angiopoietin (Ang) signaling through the Tie2 receptor regulates vasculature. The role of Ang signaling in pulmonary hypertension is well investigated, but its role in lung development is not elucidated. Here, we show that the Tie2 agonist ligand, Ang1, was detected in lung tissue at birth and its expression gradually increased in mice, whereas its antagonist Ang2 was abundant at birth and decreased inversely with Ang1. Mice expressing the potent chimeric Ang1 protein COMP-Ang1 in surfactant protein C (SPC)-positive lung epithelial cells, showed 50% lethality at birth due to respiratory failure. Surviving mice displayed impaired adaptive responsive respiratory function. Histological analysis revealed that pulmonary artery and alveolar structure were significantly dilated, and alveolar density was decreased to approximately a third of controls. Thus, the precise regulation of Tie2 signaling through an Ang1/Ang2 expression switch is important to construct a mature lung vascular network system required for normal lung development..
47. Fumio Arai, Hiroki Yoshihara, Kentaro Hosokawa, Yuka Nakamura, Yumiko Gomei, Hiroko Iwasaki, Toshio Suda, Niche regulation of hematopoietic stem cells in the endosteum
The role of thrombopoietinmpl signaling in the maintenance of quiescent Hematopoietic Stem Cells, Hematopoietic Stem Cells VII, 10.1111/j.1749-6632.2009.04561.x, 36-46, 2009.01, During postnatal life, the bone marrow (BM) supports both the self-renewal and differentiation of hematopoietic stem cells (HSCs) in specialized niches. The interaction of HSCs with their niches also regulates the quiescence of HSCs. HSC quiescence is critical to ensure lifelong hematopoiesis and to protect the HSC pool from myelotoxic insult and premature exhaustion under conditions of hematopoietic stress. Here we identified long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, Mpl, as a quiescent population in adult BM. THPO was produced by bone-lining cells in the endosteum. Inhibition and stimulation of the THPOMpl pathway produced opposite effects on the quiescence of LT-HSC. Exogenous THPO transiently increased the quiescent LT-HSC population, such as side-population and pyronin Y-negative cells. In contrast, administration of an anti-Mpl neutralizing antibody, AMM2, suppressed the quiescence of LT-HSCs and enabled HSC engraftment without irradiation, indicating that inhibition of THPOMpl signaling reduces HSC-niche interactions. Moreover, it suggests that inhibiting the HSC-niche interaction could represent a novel technique for bone marrow transplantation without irradiation. Altogether, these data suggest that the THPOMpl signaling pathway is a novel niche component in the endosteum, and in the steady-state condition, this signaling pathway plays a critical role in the regulation of LT-HSCs in the osteoblastic niche..
48. Haruko Shima, Keiyo Takubo, Hiroko Iwasaki, Hiroki Yoshihara, Yumiko Gomei, Kentaro Hosokawa, Fumio Arai, Takao Takahashi, Toshio Suda, Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2008.11.056, 378, 3, 467-472, 2009.01, Hematopoietic stem cells (HSCs) reside in hypoxic areas of the bone marrow. However, the role of hypoxia in the maintenance of HSCs has not been fully characterized. We performed xenotransplantation of human cord blood cells cultured in hypoxic or normoxic conditions into adult NOD/SCID/IL-2Rγnull (NOG) mice. Hypoxic culture (1% O2) for 6 days efficiently supported the maintenance of HSCs, although cell proliferation was suppressed compared to the normoxic culture. In contrast, hypoxia did not affect in vitro colony-forming ability. Upregulation of a cell cycle inhibitor, p21, was observed in hypoxic culture. Immunohistochemical analysis of recipient bone marrow revealed that engrafted CD34+CD38- cord blood HSCs were hypoxic. Taken together, these results demonstrate the significance of hypoxia in the maintenance of quiescent human cord blood HSCs..
49. Masako Ohmura, Kazuhito Naka, Takayuki Hoshii, Teruyuki Muraguchi, Haruhiko Shugo, Akira Tamase, Noriyuki Uema, Takako Ooshio, Fumio Arai, Keiyo Takubo, Go Nagamatsu, Isao Hamaguchi, Minoru Takagi, Masahiko Ishihara, Kazuhiro Sakurada, Hiromasa Miyaji, Toshio Suda, Atsushi Hirao, Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity, STEM CELLS, 10.1634/stemcells.2008-0506, 26, 12, 3237-3246, 2008.12, The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S-phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS-GFP Tg mice). In adult NS-GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS-GFP Tg embryos, neonates, and 10-day-old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long-term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell-cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis..
50. Sahoko Matsuoka, Yuichi Oike, Ichiro Onoyama, Atsushi Iwama, Fumio Arai, Keiyo Takubo, Yoichi Mashimo, Hideyuki Oguro, Eriko Nitta, Keisuke Ito, Kana Miyamoto, Hiroki Yoshiwara, Kentaro Hosokawa, Yuka Nakamura, Yumiko Gomei, Hiroko Iwasaki, Yasuhide Hayashi, Yumi Matsuzaki, Keiko Nakayama, Yasuo Ikeda, Akira Hata, Shigeru Chiba, Keiichi I. Nakayama, Toshio Suda, Fbxw7 acts as a critical fail-safe against premature loss of hematopoietic stem cells and development of T-ALL, Genes and Development, 10.1101/gad.1621808, 22, 8, 986-991, 2008.04, Common molecular machineries between hematopoietic stem cell (HSC) maintenance and leukemia prevention have been highlighted. The tumor suppressor Fbxw7 (F-box and WD-40 domain protein 7), a subunit of an SCF-type ubiquitin ligase complex, induces the degradation of positive regulators of the cell cycle. We demonstrate that inactivation of Fbxw7 in hematopoietic cells causes premature depletion of HSCs due to active cell cycling and p53-dependent apoptosis. Interestingly, Fbxw7 deletion also confers a selective advantage to cells with suppressed p53 function, eventually leading to development of T-cell acute lymphoblastic leukemia (TALL). Thus, Fbxw7 functions as a fail-safe mechanism against both premature HSC loss and leukemogenesis..
51. Toshio Suda, Fumio Arai, Wnt Signaling in the Niche, Cell, 10.1016/j.cell.2008.02.017, 132, 5, 729-730, 2008.03, There is much interest in understanding the signals in the bone marrow niche that keep hematopoietic stem cells (HSCs) in a quiescent state. In the current issue of Cell Stem Cell, Fleming et al. (2008) report that blocking Wnt signaling in the niche increases the number of proliferating HSCs and reduces their ability to reconstitute the hematopoietic system of irradiated recipient mice. These findings show that Wnt/β-catenin activity is crucial for the maintenance of HSC quiescence in the bone marrow niche..
52. Keiyo Takubo, Masako Ohmura, Masaki Azuma, Go Nagamatsu, Wakako Yamada, Fumio Arai, Atsushi Hirao, Toshio Suda, Stem Cell Defects in ATM-Deficient Undifferentiated Spermatogonia through DNA Damage-Induced Cell-Cycle Arrest, Cell stem cell, 10.1016/j.stem.2007.10.023, 2, 2, 170-182, 2008.02, Mammalian spermatogenesis is maintained by stem cell capacity within undifferentiated spermatogonial subpopulation. Here, using a combination of surface markers, we describe a purification method for undifferentiated spermatogonia. Flow cytometric analysis revealed that this population is composed of Plzf-positive cells and exhibits quiescence and the side population phenotype, fulfilling general stem cell criteria. We then applied this method to analyze undifferentiated spermatogonia and stem cell activity of Atm-/- mice. Atm-/- testis shows progressive depletion of undifferentiated spermatogonia accompanied by cell-cycle arrest. In Atm-/- undifferentiated spermatogonia, a self-renewal defect was observed in vitro and in vivo. Accumulation of DNA damage and activation of the p19Arf-p53-p21Cip1/Waf1 pathway were observed in Atm-/- undifferentiated spermatogonia. Moreover, suppression of p21Cip1/Waf1 in an Atm-/- background restored transplantation ability of undifferentiated spermatogonia, indicating that ATM plays an essential role in maintenance of undifferentiated spermatogonia and their stem cell capacity by suppressing DNA damage-induced cell-cycle arrest..
53. Hiroki Yoshihara, Fumio Arai, Kentaro Hosokawa, Tetsuya Hagiwara, Keiyo Takubo, Yuka Nakamura, Yumiko Gomei, Hiroko Iwasaki, Sahoko Matsuoka, Kana Miyamoto, Hiroshi Miyazaki, Takao Takahashi, Toshio Suda, Thrombopoietin/MPL Signaling Regulates Hematopoietic Stem Cell Quiescence and Interaction with the Osteoblastic Niche, Cell stem cell, 10.1016/j.stem.2007.10.020, 1, 6, 685-697, 2007.12, Maintenance of hematopoietic stem cells (HSCs) depends on interaction with their niche. Here we show that the long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, MPL, are a quiescent population in adult bone marrow (BM) and are closely associated with THPO-producing osteoblastic cells. THPO/MPL signaling upregulated β1-integrin and cyclin-dependent kinase inhibitors in HSCs. Furthermore, inhibition and stimulation of THPO/MPL pathway by treatments with anti-MPL neutralizing antibody, AMM2, and with THPO showed reciprocal regulation of quiescence of LT-HSC. AMM2 treatment reduced the number of quiescent LT-HSCs and allowed exogenous HSC engraftment without irradiation. By contrast, exogenous THPO transiently increased quiescent HSC population and subsequently induced HSC proliferation in vivo. Altogether, these observations suggest that THPO/MPL signaling plays a critical role of LT-HSC regulation in the osteoblastic niche..
54. Kentaro Hosokawa, Fumio Arai, Hiroki Yoshihara, Yuka Nakamura, Yumiko Gomei, Hiroko Iwasaki, Kana Miyamoto, Haruko Shima, Keisuke Ito, Toshio Suda, Function of oxidative stress in the regulation of hematopoietic stem cell-niche interaction, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2007.09.014, 363, 3, 578-583, 2007.11, During postnatal life, the bone marrow (BM) supports both self-renewal and differentiation of hematopoietic stem cells (HSCs) in specialized niches, such as osteoblastic niche and vascular niche. A cell adhesion molecule, N-cadherin expressed in the HSCs and osteoblasts, suggesting that homophylic binding of N-cadherin induce the adhesion of HSCs to the niche cells. Here we demonstrate that an anti-cancer drug, 5-fuluorouracil induces reactive oxygen species (ROS) in HSCs, which suppressed N-cadherin expression. These events result in the shift of side population (SP) cells to non-SP cells, indicating that quiescent HSCs are detached from the niche. Administration of a potent anti-oxidant, N-acetyl cystein (NAC) suppressed the shift from SP cells. These data suggest that ROS suppressed the N-cadherin-mediated cell adhesion, and induce the exit of HSCs from the niche..
55. Kana Miyamoto, Kiyomi Y. Araki, Kazuhito Naka, Fumio Arai, Keiyo Takubo, Satoshi Yamazaki, Sahoko Matsuoka, Takeshi Miyamoto, Keisuke Ito, Masako Ohmura, Chen Chen, Kentaro Hosokawa, Hiromitsu Nakauchi, Keiko Nakayama, Keiichi I. Nakayama, Mine Harada, Noboru Motoyama, Toshio Suda, Atsushi Hirao, Foxo3a Is Essential for Maintenance of the Hematopoietic Stem Cell Pool, Cell stem cell, 10.1016/j.stem.2007.02.001, 1, 1, 101-112, 2007.06, Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state within a bone marrow niche. Here we show that Foxo3a, a forkhead transcription factor that acts downstream of the PTEN/PI3K/Akt pathway, is critical for HSC self-renewal. We generated gene-targeted Foxo3a-/- mice and showed that, although the proliferation and differentiation of Foxo3a-/- hematopoietic progenitors were normal, the number of colony-forming cells present in long-term cocultures of Foxo3a-/- bone marrow cells and stromal cells was reduced. The ability of Foxo3a-/- HSCs to support long-term reconstitution of hematopoiesis in a competitive transplantation assay was also impaired. Foxo3a-/- HSCs also showed increased phosphorylation of p38MAPK, an elevation of ROS, defective maintenance of quiescence, and heightened sensitivity to cell-cycle-specific myelotoxic injury. Finally, HSC frequencies were significantly decreased in aged Foxo3a-/- mice compared to the littermate controls. Our results demonstrate that Foxo3a plays a pivotal role in maintaining the HSC pool..
56. Fumio Arai, Toshio Suda, Maintenance of quiescent hematopoietic stem cells in the osteoblastic niche, Hematopoietic Stem Cells VI, 10.1196/annals.1392.005, 41-53, 2007.06, Hematopoietic stem cells (HSCs) are responsible for blood cell production throughout an individual's lifetime. Interaction of HSCs with their specific microenvironments, known as stem cell niches, is critical for maintaining stem cell properties, including self-renewal capacity and the ability to differentiate into multiple lineages. During postnatal life, the bone marrow (BM) supports both self-renewal and differentiation of HSCs in specialized microenvironmental niches. In the adult BM, HSCs are located in the trabecular endosteum (osteoblastic niche) or sinusoidal perivascular (vascular niche) areas. Here we show that osteoblastic cells (OBs) are a critical component for sustaining slow-cycling or quiescent HSCs. Interaction of HSCs with OBs through signaling and cell adhesion molecules maintains the balance in HSCs between cell division/proliferation and quiescence. In particular, the quiescent state is thought to be an essential mechanism to protect HSCs from stress and to sustain long-term hematopoiesis..
57. M. Ninomiya, A. Abe, A. Katsumi, J. Xu, M. Ito, F. Arai, T. Suda, M. Ito, H. Kiyoi, T. Kinoshita, T. Naoe, Homing, proliferation and survival sites of human leukemia cells in vivo in immunodeficient mice, Leukemia, 10.1038/sj.leu.2404432, 21, 1, 136-142, 2007.04, The cellular components of the hematopoietic stem cell niche have been gradually identified. However, the niche for malignant hematopoiesis remains to be elucidated. Here, using human leukemia cells, which could be transplanted to immunodeficient mice, we studied the in vivo homing, proliferation and survival sites by immunohistopathology, compared with the corresponding sites for cord blood CD34+ (CBCD34+) cells. The human leukemia cells initially localized on the surface of osteoblasts in the epiphysial region, and expanded to the inner vascular and diaphysial regions within 4 weeks. The percentage of CD34+ leukemia cells in the bone marrow was transiently increased up to 50%. In vivo 5-bromo-2′-deoxyuridine labeling revealed that the epiphysis was the most active site for leukemia cell proliferation. CBCD34+ cells showed the similar pattern of homing and proliferation to leukemia cells. After high-dose administration of cytosine-1-β-D-arabinofuranoside, residual leukemia cells were localized in the perivascular endothelium as well as in contact with the trabecular endosteum. These findings suggest that xenotransplantation into immunodeficient mice provides a useful model to study the leukemia niche..
58. Arai Fumio, Suda Toshio, Role of Stem Cell Niche in the Maintenance of Hematopoietic Stem Cells, 炎症・再生 : 日本炎症・再生医学会雑誌 = Inflammation and regeneration, 10.2492/inflammregen.27.117, 27, 2, 117-123, 2007.03, The quiescent state in the cell cycle is thought to be indispensable for the maintenance of hematopoietic stem cells (HSCs). The interaction between HSCs and their niche is critical for maintaining the stem cell properties of HSCs, including self-renewal capacity and the ability of differentiation into single or multiple lineages. The niche cells produce signaling molecules, extracellular matrix, and cell adhesion molecules, and regulate stem cell fates. HSCs balance quiescence and cell division in the stem cell niche for long-term sustaining of hematopoiesis in the niche. Recently, long-term repopulating (LT)-HSCs exist frequently in endosteal surface of trabecular bone area in bone marrow (BM), and it was clarified that an osteoblastic cells function as a niche cells for HSCs. The specific properties of HSC are dynamically controlled by the signalings of receptor/ligand and cell adhesion molecules produced by osteoblastic cells.
We have recently reported that side-population (SP) cells in HSCs are in the quiescent state of cell cycle. Cell adhesion of HSCs to the osteoblastic niche enhanced the ability of HSCs to become quiescent, resulting in protection of the HSC compartment from stresses suppressing hematopoiesis..
59. Keisuke Ito, Keiyo Takubo, Fumio Arai, Hitoshi Satoh, Sahoko Matsuoka, Masako Ohmura, Kazuhito Naka, Masaki Azuma, Kana Miyamoto, Kentaro Hosokawa, Yasuo Ikeda, Tak W. Mak, Toshio Suda, Atsushi Hirao, Regulation of reactive oxygen species by Atm is essential for proper response to DNA double-strand breaks in lymphocytes, Journal of Immunology, 10.4049/jimmunol.178.1.103, 178, 1, 103-110, 2007.01, The ataxia telangiectasia-mutated (ATM) gene plays a pivotal role in the maintenance of genomic stability. Although it has been recently shown that antioxidative agents inhibited lymphomagenesis in Atm-/- mice, the mechanisms remain unclear. In this study, we intensively investigated the roles of reactive oxygen species (ROS) in phenotypes of Atm-/- mice. Reduction of ROS by the antioxidant N-acetyl-L-cysteine (NAC) prevented the emergence of senescent phenotypes in Atm-/- moose embryonic fibroblasts, hypersensitivity to total body irradiation, and thymic lymphomagenesis in Atm-/- mice. To understand the mechanisms for prevention of lymphomagenesis, we analyzed development of pretumor lymphocytes in Atm-/- mice. Impairment of Ig class switch recombination seen in Atm-/- mice was mitigated by NAC, indicating that ROS elevation leads to abnormal response to programmed double-strand breaks in vivo. Significantly, in vivo administration of NAC to Atm-/- mice restored normal T cell development and inhibited aberrant V(D)J recombination. We conclude that Atm-mediated ROS regulation is essential for proper DNA recombination, preventing immunodeficiency, and lymphomagenesis..
60. Keiyo Takubo, Atsushi Hirao, Masako Ohmura, Masaki Azuma, Fumio Arai, Go Nagamatsu, Toshio Suda, Premeiotic germ cell defect in seminiferous tubules of Atm-null testis, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2006.10.145, 351, 4, 993-998, 2006.12, Lifelong spermatogenesis is maintained by coordinated sequential processes including self-renewal of stem cells, proliferation of spermatogonial cells, meiotic division, and spermiogenesis. It has been shown that ataxia telangiectasia-mutated (ATM) is required for meiotic division of the seminiferous tubules. Here, we show that, in addition to its role in meiosis, ATM has a pivotal role in premeiotic germ cell maintenance. ATM is activated in premeiotic spermatogonial cells and the Atm-null testis shows progressive degeneration. In Atm-null testicular cells, differing from bone marrow cells of Atm-null mice, reactive oxygen species-mediated p16Ink4a activation does not occur in Atm-null premeiotic germ cells, which suggests the involvement of different signaling pathways from bone marrow defects. Although Atm-null bone marrow undergoes p16Ink4a-mediated cellular senescence program, Atm-null premeiotic germ cells exhibited cell cycle arrest and apoptotic elimination of premeiotic germ cells, which is different from p16Ink4a-mediated senescence..
61. Regulation of hematopoietic stem cells in the niche.
62. Keisuke Ito, Atsushi Hirao, Fumio Arai, Keiyo Takubo, Sahoko Matsuoka, Kana Miyamoto, Masako Ohmura, Kazuhito Naka, Kentaro Hosokawa, Yasuo Ikeda, Toshio Suda, Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells, Nature medicine, 10.1038/nm1388, 12, 4, 446-451, 2006.04, Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood production for their lifetime1. Appropriate control of HSC self-renewal is crucial for the maintenance of hematopoietic homeostasis. Here we show that activation of p38 MAPK in response to increasing levels of reactive oxygen species (ROS) limits the lifespan of HSCs in vivo. In Atm -/- mice, elevation of ROS levels induces HSC-specific phosphorylation of p38 MAPK accompanied by a defect in the maintenance of HSC quiescence. Inhibition of p38 MAPK rescued ROS-induced defects in HSC repopulating capacity and in the maintenance of HSC quiescence, indicating that the ROS-p38 MAPK pathway contributes to exhaustion of the stem cell population. Furthermore, prolonged treatment with an antioxidant or an inhibitor of p38 MAPK extended the lifespan of HSCs from wild-type mice in serial transplantation experiments. These data show that inactivation of p38 MAPK protects HSCs against loss of self-renewal capacity. Our characterization of molecular mechanisms that limit HSC lifespan may lead to beneficial therapies for human disease..
63. ARAI Fumio, Regulation of Hematopoietic Stem Cells and Interactions with Stem Cell Niche, Journal of oral biosciences, 10.2330/joralbiosci.48.22, 48, 1, 22-29, 2006.02, Long-term bone marrow (BM) repopulating (LTR)-hematopoietic stem cells (HSCs) exist frequently in BM trabecular bone surfaces, and it was clarified that osteoblasts (OBs) are critical for sustaining HSCs. The interaction of HSCs with their particular microenvironments, known as stem cell niches, is critical for maintaining their stem cell properties, including self-renewal capacity and the ability to differentiate into single or multiple lineages. In the niche, the fates of HSCs are regulated by the signaling molecules, extracellular matrix, and cell adhesion molecules produced by osteoblasts. Interaction of HSCs with their stem cell niches is critical for cell cycle regulation of HSCs. Especially, the quiescence of the cell cycle is thought to be an indispensable property for the maintenance of HSCs. We demonstrate that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and anti-apoptotic, transplantable and comprise a side-population (SP) of HSCs, which contact closely with angiopoietin-1 (Ang-1), a ligand for Tie2, expressing osteoblasts in the BM niche. The interaction of Tie2 and Ang-1 regulates the functional criteria of HSCs in the BM niche, including quiescence, anti-apoptosis and cell adhesion..
64. Toshio Suda, Fumio Arai, Shigeto Shimmura, Regulation of stem cells in the niche, Cornea, 24, 8 SUPPL., S12-S17, 2005.11, Stem cells have a self-renewal capacity as well as the capacity to differentiate into single or multiple lineages. In this review, we discuss the mechanism of maintenance of "sternness" in hematopoietic systems and refer to our studies of corneal epithelial stem cells. The quiescent state is believed to be indispensable for the maintenance of hematopoietic stem cells (HSC). Interaction of HSC with their particular microenvironments, known as stem cell niches, is critical for the cell cycle regulation of HSC. Monitoring of the quiescence of HSC by a side population (SP) revealed that the cell cycle status of HSC is dynamically controlled by microenvironments. We recently discovered a molecular mechanism by which the cell cycle of HSC is regulated by the niche. HSC expressing tyrosine kinase Tie2 receptors adhere to osteoblasts in the bone marrow (BM) niche. The interaction of Tie2 and its ligand angiopoietin-1 (Ang-1) leads to tight adhesion of HSC to stromal cells, resulting in the maintenance of long-term repopulating activity of HSC. Thus, the Tie2/Ang-1 signaling pathway plays a critical role in maintaining HSC in the quiescent state in the BM niche. Understanding of the relationships of stem cells to their niches should lead to development of new strategies directed toward regeneration..
65. Toshio Suda, Fumio Arai, Atsushi Hirao, Hematopoietic stem cells and their niche, Trends in Immunology, 10.1016/j.it.2005.06.006, 26, 8, 426-433, 2005.08, Recent evidence indicates that osteoblasts are crucial components of the particular microenvironments, or niches, for hematopoietic stem cells (HSCs) in adult bone marrow (BM). Stem cells persist in an immature state within the BM. The quiescence of HSCs is controlled dynamically by the signaling of receptors-ligands and cell-adhesion molecules. In this review, the characteristics of HSCs in the niche are discussed. The understanding of the relationship between normal and cancer stem cells and their niches should lead to the development of new strategies directed toward regeneration medicine and cancer therapeutics..
66. Fumio Arai, Atsushi Hirao, Toshio Suda, Regulation of hematopoietic stem cells by the niche, Trends in Cardiovascular Medicine, 10.1016/j.tcm.2005.03.002, 15, 2, 75-79, 2005.01, The quiescent state in the cell cycle is thought to be indispensable for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as niches, is critical for maintaining the stem cell properties of HSCs, including cell adhesion, survival, and cell division. Hematopoietic stem cells balance quiescence and cell division in the stem cell niche and also maintain the potential for long-term hematopoiesis. We have recently reported that HSCs expressing the receptor tyrosine kinase Tie2 are in the G0 phase and anti-apoptotic, and comprise a side-population (SP) of HSCs, which contacts osteoblasts (OBs), the source of the angiopoietin-1 (Ang-1) ligand for Tie2 in the bone marrow (BM) niche. Tie2/Ang-1 signaling occurs in interactions between HSCs and niche cells. The interaction of Tie2 with Ang-1 in vitro induces tight adhesion of HSCs to stromal cells and is sufficient to maintain the long-term blood-repopulating (LTR) activity of HSCs in vivo by preventing cell division. In addition, Ang-1 enhances the ability of HSCs to become quiescent and induces their adhesion to the bone surface in vivo, resulting in protection of the HSC compartment from stresses suppressing hematopoiesis. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in the adult BM niche. Ang-1 produced by OBs activates Tie2 on HSCs and promotes tight adhesion of HSCs to the niche, resulting in quiescence and enhanced survival of HSCs..
67. Atsushi Hirao, Fumio Arai, Toshio Suda, Regulation of cell cycle in hematopoietic stem cells by the niche, Cell Cycle, 10.4161/cc.3.12.1281, 3, 12, 1481-1483, 2004.12, The quiescent state is thought to be an indispensable property for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for cell cycle regulation of HSCs. Monitoring of the quiescence of HSCs using by a new stem cell marker, Side Population (SP), revealed that the cell cycle status of HSCs is dynamically controlled by the microenvironments. We have recently revealed a molecular mechanism in which cell cycle of HSCs is regulated by the niche. HSCs expressing the receptor tyrosine kinase Tie2 are adhere to osteoblasts (OBs) in the BM niche. The interaction of Tie2 and its ligand Angiopoietin-1 (Ang-1) leads to tight adhesion of HSCs to stromal cells, resulting in maintainance of long-term repopulating activity of HSCs. Thus, Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the BM niche. The understanding of cell cycle control in stem cells leads to development of new strategy for progress in regenerative medicine. ©2004 Landes Bioscience..
68. Keisuke Ito, Atsushi Hirao, Fumio Arai, Sahoko Matsuoka, Keiyo Takubo, Isao Hamaguchi, Kana Nomiyama, Kentaro Hosokawa, Kazuhiro Sakurada, Naomi Nakagata, Yasuo Ikeda, Tak W. Mak, Toshio Suda, Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells, Nature, 10.1038/nature02989, 431, 7011, 997-1002, 2004.10, The 'ataxia telangiectasia mutated' (Atm) gene maintains genomic stability by activating a key cell-cycle checkpoint in response to DNA damage, telomeric instability or oxidative stress. Mutational inactivation of the gene causes an autosomal recessive disorder, ataxia-telangiectasia, characterized by immunodeficiency, progressive cerebellar ataxia, oculocutaneous telangiectasia, defective spermatogenesis, premature ageing and a high incidence of lymphoma. Here we show that ATM has an essential function in the reconstitutive capacity of haematopoietic stem cells (HSCs) but is not as important for the proliferation or differentiation of progenitors, in a telomere-independent manner. Atm-/- mice older than 24 weeks showed progressive bone marrow failure resulting from a defect in HSC function that was associated with elevated reactive oxygen species. Treatment with anti-oxidative agents restored the reconstitutive capacity of Atm-/- HSCs, resulting in the prevention of bone marrow failure. Activation of the p16INK4a- retinoblastoma (Rb) gene product pathway in response to elevated reactive oxygen species led to the failure of Atm-/- HSCs. These results show that the self-renewal capacity of HSCs depends on ATM-mediated inhibition of oxidative stress..
69. Fumio Arai, Atsushi Hirao, Masako Ohmura, Hidetaka Sato, Sahoko Matsuoka, Keiyo Takubo, Keisuke Ito, Gou Young Koh, Toshio Suda, Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche, Cell, 10.1016/j.cell.2004.07.004, 118, 2, 149-161, 2004.07, The quiescent state is thought to be an indispensable property for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for adult hematopoiesis in the bone marrow (BM). Here, we demonstrate that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and antiapoptotic, and comprise a side-population (SP) of HSCs, which adhere to osteoblasts (OBs) in the BM niche. The interaction of Tie2 with its ligand Angiopoietin-1 (Ang-1) induced cobblestone formation of HSCs in vitro and maintained in vivo long-term repopulating activity of HSCs. Furthermore, Ang-1 enhanced the ability of HSCs to become quiescent and induced adhesion to bone, resulting in protection of the HSC compartment from myelosuppressive stress. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the BM niche..
70. Y Oike, Y Ito, K Yasunaga, S Matsumoto, H Maekawa, F Arai, K Hamada, K Miyata, Y Masuho, T Suda, Mast cell-released novel angiopoietin-related growth factor (AGF) promotes angiogenesis and epidermal proliferation, CIRCULATION, 106, 19, 276-277, 2002.11.
71. Yuichi Oike, Yasuhiro Ito, Koichi Hamada, Xiu Qin Zhang, Keishi Miyata, Fumio Arai, Tomohisa Inada, Kimi Araki, Naomi Nakagata, Motohiro Takeya, Yaz Y. Kisanuki, Masashi Yanagisawa, Nicholas W. Gale, Toshio Suda, Regulation of vasculogenesis and angiogenesis by EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells, Blood, 10.1182/blood.v100.4.1326.h81602001326_1326_1333, 100, 4, 1326-1333, 2002.08, Although the cellular and molecular mechanisms governing angiogenesis are only beginning to be understood, signaling through endothelial-restricted receptors, particularly receptor tyrosine kinases, has been shown to play a pivotal role in these events. Recent reports show that EphB receptor tyrosine kinases and their transmembrane-type ephrin-B2 ligands play essential roles in the embryonic vasculature. These studies suggest that cell-to-cell repellent effects due to bidirectional EphB/ephrin-B2 signaling may be crucial for vascular development, similar to the mechanism described for neuronal development. To test this hypothesis, we disrupted the precise expression pattern of EphB/ephrin-B2 in vivo by generating transgenic (CAGp-ephrin-B2 Tg) mice that express ephrin-B2 under the control of a ubiquitous and constitutive promoter, CMV enhancer-β-actin promoter-β-globin splicing acceptor (CAG). These mice displayed an abnormal segmental arrangement of intersomitic vessels, while such anomalies were not observed in Tie-2p-ephrin-B2 Tg mice in which ephrin-B2 was overexpressed in only vascular endothelial cells (ECs). This finding suggests that non-ECs expressing ephrin-B2 alter the migration of ECs expressing EphB receptors into the intersomitic region where ephrin-B2 expression is normally absent. CAGpephrin-B2 Tg mice show sudden death at neonatal stages from aortic dissecting aneurysms due to defective recruitment of vascular smooth muscle cells to the ascending aorta. EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells plays an essential role in vasculogenesis, angiogenesis, and vessel maturation..
72. Fumio Arai, Osamu Ohneda, Takeshi Miyamoto, Xiu Qin Zhang, Toshio Suda, Mesenchymal stem cells in perichondrium express activated leukocyte cell adhesion molecule and participate in bone marrow formation, Journal of Experimental Medicine, 10.1084/jem.20011700, 195, 12, 1549-1563, 2002.06, Perichondrium in fetal limb is composed of undifferentiated mesenchymal cells. However, the multipotency of cells in this region and the role of perichondrium in bone marrow formation are not well understood. In this report, we purified and characterized perichondrial cells using a monoclonal antibody against activated leukocyte cell adhesion molecule (ALCAM) and investigated the role of perichondrial cells in hematopoietic bone marrow formation. ALCAM is expressed on hematopoietic cells, endothelial cells, bone marrow stromal cells, and mesenchymal stem cells and mediates homophilic (ALCAM-ALCAM)/heterophilic (ALCAM-CD6) cell adhesion. Here we show by immunohistochemical staining that ALCAM is expressed in perichondrium. ALCAM+ perichondrial cells isolated by FACS® exhibit the characteristics of mesenchymal stem cells. ALCAM+ cells can differentiate into osteoblasts, adipocytes, chondrocytes, and stromal cells, which can support osteoclastogenesis, hematopoiesis, and angiogenesis. Furthermore, the addition of ALCAM-Fc or CD6-Fc to the metatarsal culture, the invasion of the blood vessels to a cartilage was inhibited. Our findings indicate that ALCAM+ perichondrial cells participate in vascular invasion by recruiting osteoclasts and vessels. These findings suggest that perichondrium might serve as a stem cell reservoir and play an important role in the early development of a bone and bone marrow..
73. Osamu Ohneda, Kinuko Ohneda, Fumio Arai, James Lee, Takeshi Miyamoto, Yoshimi Fukushima, Donald Dowbenko, Laurence A. Lasky, Toshio Suda, ALCAM (CD166)
Its role in hematopoietic and endothelial development, Blood, 10.1182/blood.V98.7.2134, 98, 7, 2134-2142, 2001.10, A critical role for the endothelium of yolk sac and dorsal aorta has been shown in embryonic hematopoiesis. A stromal cell line derived from yolk sac, YSCL-72, has been chosen to search for a novel molecule associated with embryonic hematopoiesis. Analysis between YSCL-72 and an adult aorta-derived endothelial cell line, EOMA, demonstrated that activated leukocyte cell adhesion molecule (ALCAM, or CD166) was specifically expressed in YSCL-72 but not in EOMA. Immunohistochemical study showed that ALCAM was expressed in the endothelium of yolk sac and dorsal aorta but not in adult aorta. ALCAM-transfected EOMA cells supported development of hematopoietic progenitor cells compared with vector-transfected EOMA cells, suggesting that ALCAM appeared to be crucial for hematopoiesis. In addition, ALCAM was found to be involved in capillary tube formation and hemangioblast differentiation. Taken together with these findings, ALCAM is highly associated not only with embryonic hematopoiesis but also vasculoangiogenesis..
74. Takeshi Miyamoto, Osamu Ohneda, Fumio Arai, Katsuya Iwamoto, Seiji Okada, Katsumasa Takagi, Dirk M. Anderson, Toshio Suda, Bifurcation of osteoclasts and dendritic cells from common progenitors, Blood, 10.1182/blood.V98.8.2544, 98, 8, 2544-2554, 2001.10, Osteoclasts and dendritic cells are derived from monocyte/macrophage precursor cells; however, how their lineage commitment is regulated is unknown. This study investigated the differentiation pathways of osteoclasts and dendritic cells from common precursor cells at the single-cell level. Osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-KB ligand (RANKL) or tumor necrosis factor-α (TNF-α) is completely inhibited by addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 at early stages of differentiation. GM-CSF-treated cells express both c-Fms and RANK and also low levels of CD11c and DEC205, which are detected on dendritic cells. Addition of GM-CSF also reduces expression of both c-Fos and Fra-1, which is an important event for inhibition of osteoclastogenesis. Overexpression of c-Fos by retroviral infection or induction in transgenic mice can rescue a failure in osteoclast differentiation even in the presence of GM-CSF. By contrast, differentiation into dendritic cells is inhibited by M-CSF, indicating that M-CSF and GM-CSF reciprocally regulate the differentia tion of both lineages. Dendritic cell maturation is also inhibited when c-Fos is expressed at an early stage of differentiation. Taken together, these findings suggest that c-Fos is a key mediator of the lineage commitment between osteoclasts and dendritic cells. The lineage determination of osteoclast progenitors seen following GM-CSF treatment functions through the regulation of c-Fos expression..
75. Takeshi Miyamoto, Fumio Arai, Osamu Ohneda, Katsumasa Takagi, Dirk M. Anderson, Toshio Suda, An adherent condition is required for formation of multinuclear osteoclasts in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor KB ligand, Blood, 10.1182/blood.v96.13.4335.h8004335_4335_4343, 96, 13, 4335-4343, 2000.12, Identification of receptor activator of nuclear factor-κB (RANK) and RANK-ligand (RANKL) has provided new insights into the osteoclast differentiation pathway. Osteoclast precursor cells were isolated using monoclonal antibodies against c-Fms and RANK, and the effect of adherence on the in vitro differentiation and proliferation of these cells was examined in 2 different types of stromal-cell-free culture systems: a semisolid culture medium (a nonadherent system) and a liquid culture medium (an adherent system). Osteoclast precursor cells were not able to differentiate into mature osteoclasts efficiently in the semisolid culture system. Trimerized RANKL enhanced osteoclast differentiation in semisolid cultures, but not to the extent seen when cells were allowed to adhere to plastic. Initial precursor cells were capable of differentiating into macrophages or osteoclasts. Once these cells were transferred to adherent conditions, striking differentiation was induced. Multinuclear cells were observed even after they had displayed phagocytic activity, which suggests that cell adhesion plays an important role in the differentiation of osteoclast precursor cells. Integrins, especially the arginineglycine-aspartic acid (RGD)-recognizing integrins αv and β3, were needed for osteoclast-committed precursor cells to proliferate in order to form multinuclear osteoclasts, and the increase in cell density affected the formation of multinuclear cells. A model of osteoclast differentiation with 2 stages of precursor development is proposed: (1) a first stage, in which precursor cells are bipotential and capable of anchorage-independent growth, and (2) a second stage, in which the further proliferation and differentiation of osteoclast-committed precursor cells is anchorage-dependent © 2000 by The American Society of Hematology..
76. Osamu Ohneda, Kinuko Ohneda, Hisayuki Nomiyama, Zhong Zheng, Steven A. Gold, Fumio Arai, Takeshi Miyamoto, Bruce E. Taillon, Richard A. McIndoe, Richard A. Shimkets, David A. Lewin, Toshio Suda, Laurence A. Lasky, WECHE: A novel hematopoietic regulatory factor, Immunity, 10.1016/s1074-7613(00)80167-3, 12, 2, 141-150, 2000.02, Previously, we described AGM-derived endothelial cell lines that either inhibited or permitted the development of erythroid or B cells. We utilized a differential gene expression method to isolate a chemokine, termed WECHE, from one of these cell lines. WECHE inhibited the formation of erythroid cells but had no effect on either myeloid or B cell formation. WECHE repressed BFU-E development from either mouse fetal liver or bone marrow progenitor cells but had no effect on colony formation induced by IL-3 or IL- 7. WECHE reduced HPP-CFC production from fetal liver-derived stem cells. WECHE hindered the growth of yolk sac-derived endothelial cells. WECHE was also chemotactic for bone marrow cells. Thus, WECHE is a novel chemokine that regulates hematopoietic differentiation..
77. Osamu Ohneda, Kinuko Ohneda, Hisayuki Nomiyama, Zhong Zheng, Steven A. Gold, Fumio Arai, Takeshi Miyamoto, Bruce E. Taillon, Richard A. McIndoe, Richard A. Shimkets, David A. Lewin, Toshio Suda, Laurence A. Lasky, WECHE
A novel hematopoietic regulatory factor, Journal of Cultural Heritage, 1, 2, 141-150, 2000.01, Previously, we described AGM-derived endothelial cell lines that either inhibited or permitted the development of erythroid or B cells. We utilized a differential gene expression method to isolate a chemokine, termed WECHE, from one of these cell lines. WECHE inhibited the formation of erythroid cells but had no effect on either myeloid or B cell formation. WECHE repressed BFU-E development from either mouse fetal liver or bone marrow progenitor cells but had no effect on colony formation induced by IL-3 or IL-7. WECHE reduced HPP-CFC production from fetal liver-derived stem cells. WECHE hindered the growth of yolk sac-derived endothelial cells. WECHE was also chemotactic for bone marrow cells. Thus, WECHE is a novel chemokine that regulates hematopoietic differentiation..
78. Fumio Arai, Takeshi Miyamoto, Osamu Ohneda, Tornohisa Inada, Tetsuo Sudo, Kenneth Brasel, Takashi Miyata, Dirk M. Anderson, Toshio Suda, Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor κB (RANK) receptors, Journal of Experimental Medicine, 10.1084/jem.190.12.1741, 190, 12, 1741-1754, 1999.12, Osteoclasts are terminally differentiated cells derived from hematopoietic stem cells. However, how their precursor cells diverge from macrophagic lineages is not known. We have identified early and late stages of osteoclastogenesis, in which precursor cells sequentially express c-Fms followed by receptor activator of nuclear factor κB (RANK), and have demonstrated that BANK expression in early-stage of precursor cells (c- Fms+RANK-) was stimulated by macrophage colony-stimulating factor (M-CSF). Although M-CSF and RANKL (ligand) induced commitment of late-stage precursor cells (c-Fms+RANK+) into osteoclasts, even late-stage precursors have the potential to differentiate into macrophages without RANKL. Pretreatment of precursors with M-CSF and delayed addition of RANKL showed that timing of RANK expression and subsequent binding of RANKL are critical for osteoclastogenesis. Thus, the RANK-RANKL system determines the osteoclast differentiation of bipotential precursors in the default pathway of macrophagic differentiation..
79. N. Kurihara, J. Tatsumi, F. Arai, A. Iwama, T. Suda, Macrophage-stimulating protein (MSP) and its receptor, RON, stimulate human osteoclast activity but not proliferation: Effect of MSP distinct from that of hepatocyte growth factor, Experimental Hematology, 26, 11, 1080-1085, 1998.10, Stem cell-derived tyrosine kinase (STK) is a member of the hepatocyte growth factor (HGF) receptor family. The ligand for STK, macrophage- stimulating protein (MSP), is a serum protein activated by members of the coagulation cascade. The RON gene is a human homolog of the murine STK. In this study we examined the role of MSP-RON in the signal pathway of human osteoclasts. Using anti-RON antibody, we detected RON expressed in multinucleated osteoclast-like cells (OCLs) formed in cultures of human bone marrow cells. To determine bone resorption, we placed OCLs on thin films of ceramic calcium phosphate formed on quartz plate-coated slides (Millenium Biologix) and measured pit formation. MSP stimulated pit formation by OCLs in a dose-dependent manner. MSP (50 ng/mL) caused a fourfold increase in pit area compared with the control. Furthermore, we examined the effects of MSP and HGF on OCL formation by purified populations of hematopoietic progenitors. OCLs were phenotypically identified by their cross-reactivity with 23c6, a monoclonal antibody that preferentially binds to osteoclasts. HGF (50 ng/mL) stimulated the differentiation of progenitors to 23c6-positive OCLs but did not enhance bone absorption. In contrast, MSP did not affect proliferation of osteoclast precursors but stimulated bone resorption by OCLs. We conclude that the MSP signal transduction pathway plays a role in bone resorption that is distinct from that of HGF..
80. KURIHARA Noriyoshi, ISHIZUKA Seiichi, TATSUMI Junichi, ARAI Fumio, IKEDA Katsumi, ROODMAN G. David, 23(S)25(R)-1,25-Dihydroxyvitamin D_3-lactone, a naturally occurring metabolite of1,25-dihydroxyvitamin D_3, inhibits osteoclast-like cell formation in human bone marrow cultures, Journal of bone and mineral metabolism, 10.1007/s007740050021, 16, 1, 5-10, 1998.08.
81. KURIHARA Noriyoshi, HOSODA Kenji, TATSUMI Junichi, YAMAJI Teizo, HOSHIHARA Eiyoshi, ARAI Fumio, IKEDA Katsumi, The N-terminal fragment of osteocalcin is released during osteoclastic bone resorption in vitro, Journal of bone and mineral metabolism, 10.1007/s007740050022, 16, 1, 11-16, 1998.08.


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