Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Fumio Arai Last modified date:2018.07.03

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


Papers
1. 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..
2. Fumio Arai, Self-renewal and differentiation of hematopoietic stem cells, [Rinsho ketsueki] The Japanese journal of clinical hematology, 57, 10, 1845-1851, 2016.01, Hematopoietic stem cells (HSCs) are characterized by their ability to self-renew and differentiate into all blood lineage cells. The fate decisions of HSCs (self-renewal versus differentiation) are made through the process of cell division and are often compared to "birth" and "death". Stem cells give rise to undifferentiated stem cells (birth) or differentiate into progenitor cells (death). This process is regulated by asymmetric/symmetric divisions of HSCs. It has been proposed that fate determination occurs as a stochastic process and that individual stem cell dynamics are randomly regulated. The behavior of HSCs is known to be regulated by the cell intrinsic factor and extrinsic (microenvironmental) stimuli. Therefore, it is possible that the signals from a specific microenvironment (niche) have the potential to control or modulate stem cell dynamics. This review focuses on the functions of the HSC niche and the application of single cell analysis for understanding the mechanisms underlying the HSC decision-making process..
3. 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..
4. 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..
5. 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..
6. 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..
7. 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..
8. 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..
9. 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..
10. 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..
11. 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..
12. 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..
13. 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..
14. Fumio Arai, Kentaro Hosokawa, Hirofumi Toyama, Yoshiko Matsumoto, Toshio Suda, Role of N-cadherin in the regulation of hematopoietic stem cells in the bone marrow niche, Annals of the New York Academy of Sciences, 10.1111/j.1749-6632.2012.06576.x, 1266, 1, 72-77, 2012.08, Cell-cell and cell-extracellular matrix interactions between hematopoietic stem cells (HSCs) and their niches are critical for the maintenance of stem cell properties. Here, it is demonstrated that a cell adhesion molecule, N-cadherin, is expressed in hematopoietic stem/progenitor cells (HSPCs) and plays a critical role in the regulation of HSPC engraftment. Furthermore, overexpression of N-cadherin in HSCs promoted quiescence and preserved HSC activity during serial bone marrow (BM) transplantation (BMT). Inhibition of N-cadherin by the transduction of N-cadherin short hairpin (sh) RNA (shN-cad) reduced the lodgment of donor HSCs to the endosteal surface, resulting in a significant reduction in long-term engraftment. shN-cad-transduced cells were maintained in the spleen for six months after BMT, indicating that N-cadherin expression in HSCs is specifically required in the BM. These findings suggest that N-cadherin-mediated cell adhesion is functionally essential for the regulation of HSPC activities in the BM niche..
15. 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..
16. 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..
17. 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, 2013.01, Prostaglandin E2 (PGE2) regulates hematopoietic stem/progenitor cell (HSPC) activity. However, the receptor(s) responsible for PGE2 signaling remains unclear. Here, we identified EP4 as a receptor activated by PGE2 to regulate HSPCs. Knockdown of Ep4 in HSPCs reduced long-term reconstitution capacity, whereas an EP4-selective agonist induced phosphorylation of GSK3β and β-catenin and enhanced long-term reconstitution capacity. Next, we analyzed the niche-mediated effect of PGE2 in HSPC regulation. Bone marrow mesenchymal progenitor cells (MPCs) expressed EP receptors, and stimulation ofMPCs with PGE2 significantly increased their ability to support HSPC colony formation. Among the EP receptor agonists, only an EP4 agonist facilitated the formation of HSPC colonies after the coculture with MPCs. PGE2 up-regulated the expression of cytokine-, cell adhesion-, extracellular matrix-, and protease-related genes in MPCs. We also examined the function of PGE2/EP4 signaling in the recovery of the HSPCs after myelosuppression. The administration of PGE2 or an EP4 agonist facilitated the recovery of HSPCs from 5-fluorouracil (5-FU)-induced myelosuppression, indicating a role for PGE2/EP4 signaling in this process. Altogether, these data suggest that EP4 is a key receptor for PGE2-mediated direct and indirect regulation of HSPCs..
18. 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..
19. 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..
20. 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..
21. Patrick S. Stumpf, Rosanna C.G. Smith, Michael Lenz, Andreas Schuppert, Franz-Josef Muller, 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..
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.
23. Matsuoka, Sahoko, Oike, Yuichi, Matsuoka, Sahoko, Oike, Yuichi, Onoyama, Ichiro, Iwama, Atsushi, Arai, Fumio, Takubo, Keiyo, Mashimo, Yoichi, Oguro, Hideyuki, Nitta, Eriko, Ito, Keisuke, Miyamoto, Kana, Yoshiwara, Hiroki, Hosokawa, Kentaro, Nakamura, Yuka, Gomei, Yumiko, Iwasaki, Hiroko, Hayashi, Yasuhide, Fbxw7 acts as a critical fail-safe against premature loss of hematopoietic stem cells and development of T-ALL, 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 (T-ALL). Thus, Fbxw7 functions as a fail-safe mechanism against both premature HSC loss and leukemogenesis..
24. Hosokawa, Kentaro, Arai, Fumio, Yoshihara, Hiroki, Iwasaki, Hiroko, Hembree, Mark, Yin, Tong, Nakamura, Yuka, Gomei, Yumiko, Takubo, Keiyo, Shiama, Haruko, Matsuoka, Sahoko, Li, Linheng, Suda, Toshio, Cadherin-Based Adhesion Is a Potential Target for Niche Manipulation to Protect Hematopoietic Stem Cells in Adult Bone Marrow, 6, 3, 197-198, 2010.03.
25. Sato, Taku, Onai, Nobuyuki, Yoshihara, Hiroki, Arai, Fumio, Suda, Toshio, Ohteki, Toshiaki, Interferon regulatory factor-2 protects quiescent hematopoietic stem cells from type I interferon-dependent exhaustion, 15, 6, 696-700, 2009.06, Type I interferons (IFNs), a family of cytokines, orchestrate numerous biological and cellular processes(1-3). Although it is well known that type I IFNs are essential for establishing the host antiviral state(4), their role in hematopoietic homeostasis has not been studied. Here we show that type I IFNs induce proliferation and exhaustion in hematopoietic stem cells (HSCs) and that interferon regulatory factor-2 (IRF2), a transcriptional suppressor of type I IFN signaling(5,6), preserves the self-renewal and multilineage differentiation capacity of HSCs. HSCs were substantially less abundant in the bone marrow of Irf2(-/-) as compared to Irf2(+/-) mice. Irf2(-/-) HSCs showed enhanced cell cycling status and failed to produce hematopoietic cells in competitive repopulation assays, and the reconstituting capacity of Irf2(-/-) HSCs was restored by disabling type I IFN signaling in these cells. In wild-type mice, injection of poly(I:C), an inducer of type I IFN signaling, or IFN-alpha induced HSC proliferation, and chronic type I IFN signaling further reduced the number of quiescent HSCs. Notably, combined poly(I:C) and 5-fluorouracil (5-FU) treatment allowed exogenous HSC engraftment and hematopoietic reconstitution in WT mice. Our findings provide insight into the molecular basis for the maintenance of HSC quiescence and may lead to improvements in bone marrow transplantation and type I IFN-based therapies for viral infection and cancer..
26. Hosokawa, Kentaro, Arai, Fumio, Yoshihara, Hiroki, Iwasaki, Hiroko, Nakamura, Yuka, Gomei, Yumiko, Suda, Toshio, Knockdown of N-cadherin suppresses the long-term engraftment of hematopoietic stem cells, 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 N-cadherin 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. .
27. Ito, Keisuke, Carracedo, Arkaitz, Weiss, Dror, Arai, Fumio, Ala, Ugo, Avigan, David E., Schafer, Zachary T., Evans, Ronald M., Suda, Toshio, Lee, Chih-Hao, Pandolfi, Pier Paolo, A PML-PPAR-delta pathway for fatty acid oxidation regulates hematopoietic stem cell maintenance, 18, 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 delta (PPAR-delta)-fatty-acid oxidation (FAO) pathway for the maintenance of hematopoietic stem cells (HSCs). We have found that loss of PPAR-delta or inhibition of mitochondrial FAO induces loss of HSC maintenance, whereas treatment with PPAR-delta agonists improved HSC maintenance. PML exerts its essential role in HSC maintenance through regulation of PPAR signaling and FAO. Mechanistically, the PML-PPAR-delta-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-delta activation increased asymmetric cell division. Thus, our findings identify a metabolic switch for the control of HSC cell fate with potential therapeutic implications..
28. Hirofumi Toyama, Fumio Arai, Kentaro Hosokawa, Yoshiko Matsumoto Ikushima, N-cadherin(+) HSCs in fetal liver exhibit higher long-term bone marrow reconstitution activity than N-cadherin(-) HSCs, 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..
29. Yoshiko Matsumoto Ikushima, Fumio Arai, Kentaro Hosokawa, Hirofumi Toyama, Keiyo Takubo, Tomoyuki Furuyashiki, Shuh Narumiya, Prostaglandin E-2 regulates murine hematopoietic stem/progenitor cells directly via EP4 receptor and indirectly through mesenchymal progenitor cells, 121, 11, 1995-2007, 2013.03, Prostaglandin E-2 (PGE(2)) regulates hematopoietic stem/progenitor cell (HSPC) activity. However, the receptor(s) responsible for PGE(2) signaling remains unclear. Here, we identified EP4 as a receptor activated by PGE(2) to regulate HSPCs. Knockdown of EP4 in HSPCs reduced long-term reconstitution capacity, whereas an EP4-selective agonist induced phosphorylation of GSK3 beta and beta-catenin and enhanced long-term reconstitution capacity. Next, we analyzed the niche-mediated effect of PGE(2) in HSPC regulation. Bone marrow mesenchymal progenitor cells (MPCs) expressed EP receptors, and stimulation of MPCs with PGE(2) significantly increased their ability to support HSPC colony formation. Among the EP receptor agonists, only an EP4 agonist facilitated the formation of HSPC colonies after the coculture with MPCs. PGE(2) up-regulated the expression of cytokine-, cell adhesion-, extracellular matrix-, and protease-related genes in MPCs. We also examined the function of PGE(2)/EP4 signaling in the recovery of the HSPCs after myelosuppression. The administration of PGE(2) or an EP4 agonist facilitated the recovery of HSPCs from 5-fluorouracil (5-FU)-induced myelosuppression, indicating a role for PGE(2)/EP4 signaling in this process. Altogether, these data suggest that EP4 is a key receptor for PGE(2)-mediated direct and indirect regulation of HSPCs..
30. Yoshiko Matsumoto Ikushima, Fumo 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, 430, 1, 20-25, 2013.01, Angiopoietin-1 (Angpt1) signaling via the Tie2 receptor regulates vascular and hematopoietic systems. To investigate the role of Angptl-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 4 week (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-Angpt1l+ (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-Kie(+) (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. (C) 2012 Elsevier Inc. All rights reserved..
31. Yuka Nakamura, Fumio Arai, Hiroko Iwasaki, Kentaro Hosokawa, Isao Kobayashi, Yumiko Gomei, Yoshiko Matsumoto, Hiroki Yoshihara, Suda Toshio, Isolation and characterization of endosteal niche cell populations that regulate hematopoietic stem cells, 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 marker(low/-) 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..
32. 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, 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/MPIL signaling upregulated beta 1-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..
33. Keisuke Ito, Atsushi Hirao, Fumio Arai, Sahoko Matsuoka, Isao Hamaguchi, Kana Nomiyama, Hosokawa Kentaro, 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, 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(1,2). 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(3,4). 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 p16(INK4a)-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..
34. Fumio Arai, Hirao Atsushi, Ohmura Masako, 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, 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..