Publications by authors named "Hitoshi Takizawa"

47 Publications

Autophagy is dispensable for the maintenance of hematopoietic stem cells in neonates.

Blood Adv 2021 Mar;5(6):1594-1604

International Research Center for Medical Sciences, Kumamoto University, Kumamoto City, Japan.

Hematopoietic stem cells (HSCs) undergo self-renewal or differentiation to sustain lifelong hematopoiesis. HSCs are preserved in quiescence with low mitochondrial activity. Recent studies indicate that autophagy contributes to HSC quiescence through suppressing mitochondrial metabolism. However, it remains unclear whether autophagy is involved in the regulation of neonatal HSCs, which proliferate actively. In this study, we clarified the role of autophagy in neonatal HSCs using 2 types of autophagy-related gene 7 (Atg7)-conditional knockout mice: Mx1-Cre inducible system and Vav-Cre system. Atg7-deficient HSCs exhibited excess cell divisions with enhanced mitochondrial metabolism, leading to bone marrow failure at adult stage. However, Atg7 deficiency minimally affected hematopoiesis and metabolic state in HSCs at neonatal stage. In addition, Atg7-deficient neonatal HSCs exhibited long-term reconstructing activity, equivalent to wild-type neonatal HSCs. Taken together, autophagy is dispensable for stem cell function and hematopoietic homeostasis in neonates and provide a novel aspect into the role of autophagy in the HSC regulation.
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http://dx.doi.org/10.1182/bloodadvances.2020002410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7993101PMC
March 2021

Immuno-Modulation of Hematopoietic Stem and Progenitor Cells in Inflammation.

Front Immunol 2020 24;11:585367. Epub 2020 Nov 24.

Laboratory of Stem Cell Stress, International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan.

Lifelong blood production is maintained by bone marrow (BM)-residing hematopoietic stem cells (HSCs) that are defined by two special properties: multipotency and self-renewal. Since dysregulation of either may lead to a differentiation block or extensive proliferation causing dysplasia or neoplasia, the genomic integrity and cellular function of HSCs must be tightly controlled and preserved by cell-intrinsic programs and cell-extrinsic environmental factors of the BM. The BM had been long regarded an immune-privileged organ shielded from immune insults and inflammation, and was thereby assumed to provide HSCs and immune cells with a protective environment to ensure blood and immune homeostasis. Recently, accumulating evidence suggests that hemato-immune challenges such as autoimmunity, inflammation or infection elicit a broad spectrum of immunological reactions in the BM, and in turn, influence the function of HSCs and BM environmental cells. Moreover, in analogy with the emerging concept of "trained immunity", certain infection-associated stimuli are able to train HSCs and progenitors to produce mature immune cells with enhanced responsiveness to subsequent challenges, and in some cases, form an inflammatory or infectious memory in HSCs themselves. In this review, we will introduce recent findings on HSC and hematopoietic regulation upon exposure to various hemato-immune stimuli and discuss how these challenges can elicit either beneficial or detrimental outcomes on HSCs and the hemato-immune system, as well as their relevance to aging and hematologic malignancies.
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http://dx.doi.org/10.3389/fimmu.2020.585367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732516PMC
November 2020

Establishment of bone marrow-derived M-CSF receptor-dependent self-renewing macrophages.

Cell Death Discov 2020 23;6:63. Epub 2020 Jul 23.

Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto, 860-0811 Japan.

Recent studies have revealed that tissue macrophages are derived from yolk sac precursors or fetal liver monocytes, in addition to bone marrow monocytes. The relative contribution of these cells to the tissue macrophage pool is not fully understood, but embryo-derived cells are supposed to be more important because of their capacity to self-renew. Here, we show the presence of adult bone marrow-derived macrophages that retain self-renewing capacity. The self-renewing macrophages were readily obtained by long-term culture of mouse bone marrow cells with macrophage colony-stimulating factor (M-CSF), a key cytokine for macrophage development. They were non-tumorigenic and proliferated in the presence of M-CSF in unlimited numbers. Despite several differences from non-proliferating macrophages, they retained many features of cells of the monocytic lineage, including the differentiation into dendritic cells or osteoclasts. Among the transcription factors involved in the self-renewal of embryonic stem cells, Krüppel-like factor 2 (KLF2) was strongly upregulated upon M-CSF stimulation in the self-renewing macrophages, which was accompanied by the downregulation of MafB, a transcription factor that suppresses KLF2 expression. Indeed, knockdown of KLF2 led to cell cycle arrest and diminished cell proliferation in the self-renewing macrophages. Our new cell model would be useful to unravel differences in phenotype, function, and molecular mechanism of proliferation among self-renewing macrophages with different origins.
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http://dx.doi.org/10.1038/s41420-020-00300-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378060PMC
July 2020

[Innate immune signal-mediated regulation and alterations in hematopoiesis].

Rinsho Ketsueki 2020 ;61(6):651-656

International Research Center for Medical Sciences, Kumamoto University.

Hematopoietic stem cells (HSCs) are maintained in steady state in the bone marrow (BM); these cells are capable of continuous self-renewal and have the potential for multilineage-differentiation into all blood cell lineages. The BM has long been considered as an immune-privilege organ with little immunological reactions. However, recent findings have revealed that immunological/hematopoietic challenges such as infection or inflammation induce broad spectrum of immune and inflammatory responses in BM. While these responses play a beneficial role to boost immune activation and blood production, chronic challenge might lead to BM pathology and dysregulation, including hematopoietic aplasia or neoplasia. We will introduce recent findings focused on hematopoietic activation induced by existing outside of our body or co-existing with us, and discuss to what degree and how function of HSCs and progenitors is regulated and altered by bacterial insult.
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http://dx.doi.org/10.11406/rinketsu.61.651DOI Listing
July 2020

Genetic fingerprint defines hematopoietic stem cell pool size and function.

Haematologica 2020 03;105(3):526-528

Laboratory of Stem Cell Stress, International Research Center for Medical Sciences, Kumamoto University

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http://dx.doi.org/10.3324/haematol.2019.241299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049336PMC
March 2020

iPSC-Derived Platelets Depleted of HLA Class I Are Inert to Anti-HLA Class I and Natural Killer Cell Immunity.

Stem Cell Reports 2020 01 26;14(1):49-59. Epub 2019 Dec 26.

Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan. Electronic address:

The ex vivo production of platelets depleted of human leukocyte antigen class I (HLA-I) could serve as a universal measure to overcome platelet transfusion refractoriness caused by HLA-I incompatibility. Here, we developed human induced pluripotent cell-derived HLA-I-deficient platelets (HLA-KO iPLATs) in a clinically applicable imMKCL system by genetic manipulation and assessed their immunogenic properties including natural killer (NK) cells, which reject HLA-I downregulated cells. HLA-KO iPLATs were deficient for all HLA-I but did not elicit a cytotoxic response by NK cells in vitro and showed circulation equal to wild-type iPLATs upon transfusion in our newly established Hu-NK-MSTRG mice reconstituted with human NK cells. Additionally, HLA-KO iPLATs successfully circulated in an alloimmune platelet transfusion refractoriness model of Hu-NK-MISTRG mice. Mechanistically, the lack of NK cell-activating ligands on platelets may be responsible for evading the NK cell response. This study revealed the unique non-immunogenic property of platelets and provides a proof of concept for the clinical application of HLA-KO iPLATs.
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http://dx.doi.org/10.1016/j.stemcr.2019.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962657PMC
January 2020

Discrimination of Dormant and Active Hematopoietic Stem Cells by G Marker Reveals Dormancy Regulation by Cytoplasmic Calcium.

Cell Rep 2019 12;29(12):4144-4158.e7

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between "dormant" and "active" HSCs remains unresolved. Here we generate a G marker (GM) mouse line that visualizes quiescent cells and identify a small population of active HSCs (GM), which are distinct from dormant HSCs (GM), within the conventional quiescent HSC fraction. Single-cell RNA-seq analyses show that the gene expression profiles of these populations are nearly identical but differ in their Cdk4/6 activity. Furthermore, high-throughput small-molecule screening reveals that high concentrations of cytoplasmic calcium ([Ca]) are linked to dormancy of HSCs. These findings indicate that GM separates dormant and active adult HSCs, which are regulated by Cdk4/6 and [Ca]. This GM mouse line represents a useful resource for investigating physiologically important stem cell subpopulations.
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http://dx.doi.org/10.1016/j.celrep.2019.11.061DOI Listing
December 2019

Antitumor immunity augments the therapeutic effects of p53 activation on acute myeloid leukemia.

Nat Commun 2019 10 25;10(1):4869. Epub 2019 Oct 25.

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

The negative regulator of p53, MDM2, is frequently overexpressed in acute myeloid leukemia (AML) that retains wild-type TP53 alleles. Targeting of p53-MDM2 interaction to reactivate p53 function is therefore an attractive therapeutic approach for AML. Here we show that an orally active inhibitor of p53-MDM2 interaction, DS-5272, causes dramatic tumor regressions of MLL-AF9-driven AML in vivo with a tolerable toxicity. However, the antileukemia effect of DS-5272 is markedly attenuated in immunodeficient mice, indicating the critical impact of systemic immune responses that drive p53-mediated leukemia suppression. In relation to this, DS-5272 triggers immune-inflammatory responses in MLL-AF9 cells including upregulation of Hif1α and PD-L1, and inhibition of the Hif1α-PD-L1 axis sensitizes AML cells to p53 activation. We also found that NK cells are important mediators of antileukemia immunity. Our study showed the potent activity of a p53-activating drug against AML, which is further augmented by antitumor immunity.
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http://dx.doi.org/10.1038/s41467-019-12555-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814808PMC
October 2019

Fate Distribution and Regulatory Role of Human Mesenchymal Stromal Cells in Engineered Hematopoietic Bone Organs.

iScience 2019 Sep 7;19:504-513. Epub 2019 Aug 7.

Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland. Electronic address:

The generation of humanized ectopic ossicles (hOss) in mice has been proposed as an advanced translational and fundamental model to study the human hematopoietic system. The approach relies on the presence of human bone marrow-derived mesenchymal stromal cells (hMSCs) supporting the engraftment of transplanted human hematopoietic stem and progenitor cells (HSPCs). However, the functional distribution of hMSCs within the humanized microenvironment remains to be investigated. Here, we combined genetic tools and quantitative confocal microscopy to engineer and subsequently analyze hMSCs' fate and distribution in hOss. Implanted hMSCs reconstituted a humanized environment including osteocytes, osteoblasts, adipocytes, and stromal cells associated with vessels. By imaging full hOss, we identified rare physical interactions between hMSCs and human CD45+/CD34+/CD90+ cells, supporting a functional contact-triggered regulatory role of hMSCs. Our study highlights the importance of compiling quantitative information from humanized organs, to decode the interactions between the hematopoietic and the stromal compartments.
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http://dx.doi.org/10.1016/j.isci.2019.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6710718PMC
September 2019

Hlf marks the developmental pathway for hematopoietic stem cells but not for erythro-myeloid progenitors.

J Exp Med 2019 07 10;216(7):1599-1614. Epub 2019 May 10.

Department of Hematology, Juntendo University Graduate School of Medicine, Tokyo, Japan.

Before the emergence of hematopoietic stem cells (HSCs), lineage-restricted progenitors, such as erythro-myeloid progenitors (EMPs), are detected in the embryo or in pluripotent stem cell cultures in vitro. Although both HSCs and EMPs are derived from hemogenic endothelium, it remains unclear how and when these two developmental programs are segregated during ontogeny. Here, we show that hepatic leukemia factor (Hlf) expression specifically marks a developmental continuum between HSC precursors and HSCs. Using the -tdTomato reporter mouse, we found that is expressed in intra-aortic hematopoietic clusters and fetal liver HSCs. In contrast, EMPs and yolk sac hematopoietic clusters before embryonic day 9.5 do not express HSC specification, regulated by the Evi-1/Hlf axis, is activated only within Hlf nascent hematopoietic clusters. These results strongly suggest that HSCs and EMPs are generated from distinct cohorts of hemogenic endothelium. Selective induction of the Hlf lineage pathway may lead to the in vitro generation of HSCs from pluripotent stem cells.
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http://dx.doi.org/10.1084/jem.20181399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605751PMC
July 2019

Development of the hematopoietic system: Role of inflammatory factors.

Wiley Interdiscip Rev Dev Biol 2019 07 27;8(4):e341. Epub 2019 Mar 27.

International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan.

Hematopoietic stem cells (HSCs) have two defining features, multipotency and self-renewal, both of which are tightly controlled by cell autonomous programs and environmental factors throughout the lifetime of an organism. During development, HSCs are born in the aorta-gonad-mesonephros region, and migrate to distinct hematopoietic organs such as the placenta, fetal liver and spleen, continuously self-renewing and expanding to reach a homeostatic number. HSCs ultimately seed the bone marrow around the time of birth and become dormant to sustain lifelong hematopoiesis. In this review, we will summarize the recent findings on the role of inflammatory factors regulating HSC development, that is, emergence, trafficking and differentiation. An understanding of HSC kinetics during developmental processes will provide useful knowledge on HSC behavior under physiological and pathophysiological conditions. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Adult Stem Cells, Tissue Renewal, and Regeneration > Environmental Control of Stem Cells.
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http://dx.doi.org/10.1002/wdev.341DOI Listing
July 2019

Erratum to "Engineered humanized bone organs maintain human hematopoiesis in vivo": [Experimental Hematology 61 (2018) 45-51].

Exp Hematol 2019 Apr 26;72:72. Epub 2019 Jan 26.

Hematology, University Hospital Zurich and University of Zurich, Zurich, Switzerland; International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan.

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http://dx.doi.org/10.1016/j.exphem.2019.01.007DOI Listing
April 2019

Thrombopoietin Metabolically Primes Hematopoietic Stem Cells to Megakaryocyte-Lineage Differentiation.

Cell Rep 2018 11;25(7):1772-1785.e6

Cancer Science Institute, National University of Singapore, 14 Medical Drive, MD6, 117599 Singapore, Singapore; International Research Center for Medical Sciences, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto City 860-0811, Japan. Electronic address:

During acute myelosuppression or thrombocytopenia, bone marrow (BM) hematopoietic cells respond rapidly to replenish peripheral blood platelets. While the cytokine thrombopoietin (Thpo) both regulates platelet production and maintains HSC potential, whether Thpo controls megakaryocyte (Mk)-lineage differentiation of HSCs is unclear. Here, we show that Thpo rapidly upregulates mitochondrial activity in HSCs, an activity accompanied by differentiation to an Mk lineage. Moreover, in unperturbed hematopoiesis, HSCs with high mitochondrial activity exhibit Mk-lineage differentiation in vitro and myeloid lineage-biased reconstitution in vivo. Furthermore, Thpo skewed HSCs to express the tetraspanin CD9, a pattern correlated with mitochondrial activity. Mitochondria-active HSCs are resistant to apoptosis and oxidative stress upon Thpo stimulation. Thpo-regulated mitochondrial activity associated with mitochondrial translocation of STAT3 phosphorylated at serine 727. Overall, we report an important role for Thpo in regulating rapid Mk-lineage commitment. Thpo-dependent changes in mitochondrial metabolism prime HSCs to undergo direct differentiation to an Mk lineage.
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http://dx.doi.org/10.1016/j.celrep.2018.10.059DOI Listing
November 2018

[Inflammation and early hematopoiesis].

Rinsho Ketsueki 2018;59(10):1955-1961

International Research Center for Medical Sciences, Kumamoto University.

Lifelong, self-renewing, and, multilineage-differentiating hematopoietic stem cells (HSCs) gradually divide in steady-state bone marrow (BM). Conversely, in cases of hematopoietic stress, including infection and inflammation, hematopoiesis is highly demanded due to massive cell consumption in the stressed tissues and involves HSC recruitment to fulfil the hematopoietic demand. Accumulating evidence indicates that infection-related inflammation acts on blood-forming HSCs and progenitors within the BM to facilitate hematopoiesis for self-defense. In this review, we discuss the mechanisms used by various inflammatory responses involving not only HSCs but also the niche cells in the BM, a site that has long been considered an immune-privileged organ.
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http://dx.doi.org/10.11406/rinketsu.59.1955DOI Listing
July 2019

Engineered humanized bone organs maintain human hematopoiesis in vivo.

Exp Hematol 2018 05 9;61:45-51.e5. Epub 2018 Feb 9.

Hematology, University Hospital Zurich and University of Zurich, Zurich, Switzerland; International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan. Electronic address:

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) environment, the so-called HSC niche, that provides pivotal factors for their maintenance. Although the cellular and molecular components of the mouse BM HSC niche have been extensively studied using genetically modified animals, relatively little is known about the counterpart human BM niche components. We previously illustrated, with a developmental tissue engineering approach, that human adult BM-derived mesenchymal stromal cells (MSCs) can develop into human bone organs (so-called ossicles) through endochondral ossification in vivo and that these human ossicles are able to maintain functional mouse HSCs. We here report that human ossicles in immunodeficient mice maintain human immature and mature hematopoiesis in vivo. Moreover, a higher percentage of human stem and progenitor cells are kept in quiescence in human ossicles as compared with mouse BM. These findings indicate that the human MSC-derived ossicles function as a hematopoietic niche and may potentially serve as a re-engineerable platform to study normal and diseased human hematopoiesis in a physiologically optimized environment.
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http://dx.doi.org/10.1016/j.exphem.2018.01.004DOI Listing
May 2018

Pathogen-Induced TLR4-TRIF Innate Immune Signaling in Hematopoietic Stem Cells Promotes Proliferation but Reduces Competitive Fitness.

Cell Stem Cell 2017 08 20;21(2):225-240.e5. Epub 2017 Jul 20.

Hematology, University Hospital Zurich and University of Zurich, 8091 Zurich, Switzerland. Electronic address:

Bacterial infection leads to consumption of short-lived innate immune effector cells, which then need to be replenished from hematopoietic stem and progenitor cells (HSPCs). HSPCs express pattern recognition receptors, such as Toll-like receptors (TLRs), and ligation of these receptors induces HSPC mobilization, cytokine production, and myeloid differentiation. The underlying mechanisms involved in pathogen signal transduction in HSCs and the resulting biological consequences remain poorly defined. Here, we show that in vivo lipopolysaccharide (LPS) application induces proliferation of dormant HSCs directly via TLR4 and that sustained LPS exposure impairs HSC self-renewal and competitive repopulation activity. This process is mediated via TLR4-TRIF-ROS-p38, but not MyD88 signaling, and can be inhibited pharmacologically without preventing emergency granulopoiesis. Live Salmonella Typhimurium infection similarly induces proliferative stress in HSCs, in part via TLR4-TRIF signals. Thus, while direct TLR4 activation in HSCs might be beneficial for controlling systemic infection, prolonged TLR4 signaling has detrimental effects and may contribute to inflammation-associated HSPC dysfunction.
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http://dx.doi.org/10.1016/j.stem.2017.06.013DOI Listing
August 2017

Impact of inflammation on early hematopoiesis and the microenvironment.

Int J Hematol 2017 Jul 30;106(1):27-33. Epub 2017 May 30.

Division of Hematology, University Hospital Zurich and University of Zurich, CH-8091, Zurich, Switzerland.

Steady-state hematopoiesis is maintained by slowly dividing, self-renewing hematopoietic stem cells (HSCs) and their offspring, lineage-specified downstream progenitors in bone marrow (BM). It was previously thought that hematopoietic stresses such as infection or other inflammatory stimuli, are mostly recognized by terminally differentiated immune cells, i.e., front-line defenders at the local site of reaction, and that they produce factors that directly act on hematopoietic stem and progenitors (HSPCs) in BM and subsequently stimulate them to rebuild and sustain the hemato-lymphatic system. However, accumulating evidence now indicates that primitive HSPCs, as well as microenvironmental cells in BM are also able to sense systemically migrating hematopoietic stress signals, and respond by orchestrating on-site hematopoiesis via direct and indirect mechanisms. While inflammation has many beneficial roles in activating the immune system for defense or facilitating tissue repair, it also shows detrimental effects if sustained chronically, i.e., might lead to HSPC damage as bone marrow failure or leukemia. Thus, inflammation requires tight control of initiation and termination in time and space dependent manner.
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http://dx.doi.org/10.1007/s12185-017-2266-5DOI Listing
July 2017

Inflamm-Aging of Hematopoiesis, Hematopoietic Stem Cells, and the Bone Marrow Microenvironment.

Front Immunol 2016 14;7:502. Epub 2016 Nov 14.

International Research Center for Medical Sciences , Kumamoto , Japan.

All hematopoietic and immune cells are continuously generated by hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) through highly organized process of stepwise lineage commitment. In the steady state, HSCs are mostly quiescent, while HPCs are actively proliferating and contributing to daily hematopoiesis. In response to hematopoietic challenges, e.g., life-threatening blood loss, infection, and inflammation, HSCs can be activated to proliferate and engage in blood formation. The HSC activation induced by hematopoietic demand is mediated by direct or indirect sensing mechanisms involving pattern recognition receptors or cytokine/chemokine receptors. In contrast to the hematopoietic challenges with obvious clinical symptoms, how the aging process, which involves low-grade chronic inflammation, impacts hematopoiesis remains undefined. Herein, we summarize recent findings pertaining to functional alternations of hematopoiesis, HSCs, and the bone marrow (BM) microenvironment during the processes of aging and inflammation and highlight some common cellular and molecular changes during the processes that influence hematopoiesis and its cells of origin, HSCs and HPCs, as well as the BM microenvironment. We also discuss how age-dependent alterations of the immune system lead to subclinical inflammatory states and how inflammatory signaling might be involved in hematopoietic aging. Our aim is to present evidence supporting the concept of "Inflamm-Aging," or inflammation-associated aging of hematopoiesis.
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http://dx.doi.org/10.3389/fimmu.2016.00502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107568PMC
November 2016

MPL expression on AML blasts predicts peripheral blood neutropenia and thrombocytopenia.

Blood 2016 11 29;128(18):2253-2257. Epub 2016 Aug 29.

Hematology, University Hospital and University of Zurich, Zurich, Switzerland.

Although the molecular pathways that cause acute myeloid leukemia (AML) are increasingly well understood, the pathogenesis of peripheral blood cytopenia, a major cause of AML mortality, remains obscure. A prevailing assumption states that AML spatially displaces nonleukemic hematopoiesis from the bone marrow. However, examining an initial cohort of 223 AML patients, we found no correlation between bone marrow blast content and cytopenia, questioning the displacement theory. Measuring serum concentration of thrombopoietin (TPO), a key regulator of hematopoietic stem cells and megakaryocytes, revealed loss of physiologic negative correlation with platelet count in AML cases with blasts expressing MPL, the thrombopoietin (scavenging) receptor. Mechanistic studies demonstrated that MPL blasts could indeed clear TPO, likely therefore leading to insufficient cytokine levels for nonleukemic hematopoiesis. Microarray analysis in an independent multicenter study cohort of 437 AML cases validated MPL expression as a central predictor of thrombocytopenia and neutropenia in AML. Moreover, t(8;21) AML cases demonstrated the highest average MPL expression and lowest average platelet and absolute neutrophil counts among subgroups. Our work thus explains the pathophysiology of peripheral blood cytopenia in a relevant number of AML cases.
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http://dx.doi.org/10.1182/blood-2016-04-711986DOI Listing
November 2016

Hematopoietic stem cell response to inflammation.

Authors:
Hitoshi Takizawa

Rinsho Ketsueki 2016 08;57(8):1059-65

International Research Center for Medical Sciences, Kumamoto University.

Hematopoietic stem cells (HSCs) have unique functional properties, including self-renewal and multi-lineage differentiation potential, and are thought to be fully responsible for lifelong hematopoiesis. However, recent studies have shown that HSCs divide much more slowly than thought, and, therefore, that daily hematopoiesis is maintained not by HSCs but by hematopoietic progenitors with limited self-renewal. When hematopoietic stress such as an infection occurs, hematopoietic production is at high demand at the site of infection. To meet hematopoietic needs, HSCs are also presumably recruited to orchestrate hematopoiesis. The beneficial and detrimental effects of inflammation on HSC function and the associated hematopoietic regulation are discussed herein, by summarizing recent findings.
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http://dx.doi.org/10.11406/rinketsu.57.1059DOI Listing
August 2016

Enhanced thrombopoietin but not G-CSF receptor stimulation induces self-renewing hematopoietic stem cell divisions in vivo.

Blood 2016 06 4;127(25):3175-9. Epub 2016 May 4.

Division of Hematology, University Hospital Zurich and University of Zurich, Zurich, Switzerland; and International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan.

In steady-state adult hematopoiesis, most hematopoietic stem cells (HSCs) are in the resting phase of the cell cycle. Upon enhanced hematopoietic demand, HSCs can be induced to divide and self-renew or differentiate. However, the cell-extrinsic signals inducing HSC cycling remain to be elucidated. Using in vivo high-resolution single HSC divisional tracking, we directly demonstrate that clinically applied thrombopoietin receptor but not granulocyte colony-stimulating factor (G-CSF) receptor agonists drive HSCs into self-renewing divisions leading to quantitative expansion of functional HSC as defined by their in vivo serial multilineage and long-term repopulating potential. These results suggest that thrombopoietin mimetics might be applicable to expand HSCs in vivo and to sensitize thrombopoietin receptor-expressing HSCs to cell cycle-dependent cytotoxic agents.
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http://dx.doi.org/10.1182/blood-2015-09-669929DOI Listing
June 2016

LPS-stimulated human bone marrow stroma cells support myeloid cell development and progenitor cell maintenance.

Ann Hematol 2016 Jan 11;95(2):173-8. Epub 2015 Nov 11.

Department of Oncology, Hematology, Hemostaseology and Stem cell transplantation, University Hospital Aachen, RWTH Aachen University, Pauwelsstrasse 30, 52074, Aachen, Germany.

The nonhematopoietic bone marrow (BM) microenvironment provides a functional niche for hematopoietic cell maintenance, recruitment, and differentiation. It consists of multiple cell types including vasculature, bone, adipose tissue, and fibroblast-like bone marrow stromal cells (BMSC), which can be summarized under the generic term niche cells. BMSC express Toll-like receptors (TLRs) and are capable to respond to TLR-agonists by changing their cytokine expression pattern in order to more efficiently support hematopoiesis. Here, we show that in addition to enhanced myeloid colony formation from human CD34+ cells, lipopolysaccharide (LPS) stimulation retains overall higher numbers of CD34+ cells in co-culture assays using BMSC, with eightfold more CD34+ cells that underwent up to three divisions as compared to non-stimulated assays. When subjected to cytokine-supplemented myeloid colony-forming unit (CFU) assays or transplanted into newborn RAG2(-/-) γc (-/-) mice, CD34(+) cells from LPS-stimulated BMSC cultures give rise to the full spectrum of myeloid colonies and T and B cells, respectively, thus supporting maintenance of myeloid and lymphoid primed hematopoietic progenitor cells (HPCs) under inflammatory conditions. Collectively, we suggest that BMSC enhance hematopoiesis during inflammatory conditions to support the replenishment of innate immune effector cells and to prevent the exhaustion of the hematopoietic stem and progenitor cell (HSPC) pool.
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http://dx.doi.org/10.1007/s00277-015-2550-5DOI Listing
January 2016

The analysis, roles and regulation of quiescence in hematopoietic stem cells.

Development 2014 Dec;141(24):4656-66

Department of Cell Differentiation, The Sakaguchi Laboratory, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan Cancer Science Institute, National University of Singapore, 14 Medical Drive MD6, Centre for Translational Medicine, 117599 Singapore

Tissue homeostasis requires the presence of multipotent adult stem cells that are capable of efficient self-renewal and differentiation; some of these have been shown to exist in a dormant, or quiescent, cell cycle state. Such quiescence has been proposed as a fundamental property of hematopoietic stem cells (HSCs) in the adult bone marrow, acting to protect HSCs from functional exhaustion and cellular insults to enable lifelong hematopoietic cell production. Recent studies have demonstrated that HSC quiescence is regulated by a complex network of cell-intrinsic and -extrinsic factors. In addition, detailed single-cell analyses and novel imaging techniques have identified functional heterogeneity within quiescent HSC populations and have begun to delineate the topological organization of quiescent HSCs. Here, we review the current methods available to measure quiescence in HSCs and discuss the roles of HSC quiescence and the various mechanisms by which HSC quiescence is maintained.
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http://dx.doi.org/10.1242/dev.106575DOI Listing
December 2014

Myeloproliferative neoplasms can be initiated from a single hematopoietic stem cell expressing JAK2-V617F.

J Exp Med 2014 Oct 6;211(11):2213-30. Epub 2014 Oct 6.

Department of Biomedicine, Experimental Hematology, University Hospital Basel and University of Basel, 4031 Basel, Switzerland

The majority of patients with myeloproliferative neoplasms (MPNs) carry a somatic JAK2-V617F mutation. Because additional mutations can precede JAK2-V617F, it is questioned whether JAK2-V617F alone can initiate MPN. Several mouse models have demonstrated that JAK2-V617F can cause MPN; however, in all these models disease was polyclonal. Conversely, cancer initiates at the single cell level, but attempts to recapitulate single-cell disease initiation in mice have thus far failed. We demonstrate by limiting dilution and single-cell transplantations that MPN disease, manifesting either as erythrocytosis or thrombocytosis, can be initiated clonally from a single cell carrying JAK2-V617F. However, only a subset of mice reconstituted from single hematopoietic stem cells (HSCs) displayed MPN phenotype. Expression of JAK2-V617F in HSCs promoted cell division and increased DNA damage. Higher JAK2-V617F expression correlated with a short-term HSC signature and increased myeloid bias in single-cell gene expression analyses. Lower JAK2-V617F expression in progenitor and stem cells was associated with the capacity to stably engraft in secondary recipients. Furthermore, long-term repopulating capacity was also present in a compartment with intermediate expression levels of lineage markers. Our studies demonstrate that MPN can be initiated from a single HSC and illustrate that JAK2-V617F has complex effects on HSC biology.
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http://dx.doi.org/10.1084/jem.20131371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203945PMC
October 2014

BRAF-V600E expression in precursor versus differentiated dendritic cells defines clinically distinct LCH risk groups.

J Exp Med 2014 Apr 17;211(4):669-83. Epub 2014 Mar 17.

Department of Oncological Sciences, 2 Tisch Cancer Institute, and 3 Immunology Institute, Mount Sinai School of Medicine, New York, NY 10029.

Langerhans cell histiocytosis (LCH) is a clonal disorder with elusive etiology, characterized by the accumulation of CD207(+) dendritic cells (DCs) in inflammatory lesions. Recurrent BRAF-V600E mutations have been reported in LCH. In this study, lesions from 100 patients were genotyped, and 64% carried the BRAF-V600E mutation within infiltrating CD207(+) DCs. BRAF-V600E expression in tissue DCs did not define specific clinical risk groups but was associated with increased risk of recurrence. Strikingly, we found that patients with active, high-risk LCH also carried BRAF-V600E in circulating CD11c(+) and CD14(+) fractions and in bone marrow (BM) CD34(+) hematopoietic cell progenitors, whereas the mutation was restricted to lesional CD207(+) DC in low-risk LCH patients. Importantly, BRAF-V600E expression in DCs was sufficient to drive LCH-like disease in mice. Consistent with our findings in humans, expression of BRAF-V600E in BM DC progenitors recapitulated many features of the human high-risk LCH, whereas BRAF-V600E expression in differentiated DCs more closely resembled low-risk LCH. We therefore propose classification of LCH as a myeloid neoplasia and hypothesize that high-risk LCH arises from somatic mutation of a hematopoietic progenitor, whereas low-risk disease arises from somatic mutation of tissue-restricted precursor DCs.
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http://dx.doi.org/10.1084/jem.20130977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3978272PMC
April 2014

A comprehensive surface proteome analysis of myeloid leukemia cell lines for therapeutic antibody development.

J Proteomics 2014 Mar 30;99:138-51. Epub 2014 Jan 30.

ETH Zurich, Department of Chemistry and Applied Biosciences, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland. Electronic address:

Unlabelled: A detailed characterization of the cell surface proteome facilitates the identification of target antigens, which can be used for the development of antibody-based therapeutics for the treatment of hematological malignancies. We have performed cell surface biotinylation of five human myeloid leukemia cell lines and normal human granulocytes, which was used for mass spectrometric analysis and allowed the identification and label-free, relative quantification of 320 membrane proteins. Several proteins exhibited a pronounced difference in expression between leukemia cell lines and granulocytes. We focused our attention on CD166/ALCAM, as this protein was strongly up-regulated on all AML cell lines and AML blasts of some patients. A human monoclonal antibody specific to CD166 (named H8) was generated using phage display technology. H8 specifically recognized AML cells in FACS analysis while demonstrating tumor targeting properties in vivo. After in vitro screening of five potent cytotoxic agents, a duocarmycin derivative was used for the preparation of an antibody-drug conjugate, which was able to kill AML cells in vitro with an IC50 of 8nM. The presented atlas of surface proteins in myeloid leukemia provides an experimental basis for the choice of target antigens, which may be used for the development of anti-AML therapeutic antibodies.

Biological Significance: The ability to discriminate between malignant and healthy, essential cells represents an important requirement for the development of armed antibodies for the therapy of hematological malignancies. Our proteomic study is, to our knowledge, the first large scale comparison of the accessible cell surface proteome of leukemia cells and normal blood cells, facilitating the choice of a suitable target for the treatment of acute myeloid leukemia (AML). An antibody drug conjugate was generated recognizing the CD166 antigen which was found to be strongly up-regulated in all AML cell lines and AML blasts of some patients. This antibody drug conjugate SIP(H8)-Duo might be further characterized in therapy experiments and might lead to a new targeted treatment option for AML.
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http://dx.doi.org/10.1016/j.jprot.2014.01.022DOI Listing
March 2014

Fas (CD95) expression in myeloid cells promotes obesity‐induced muscle insulin resistance.

EMBO Mol Med 2014 01;6(1):43-56

Low-grade inflammation in adipose tissue and liver has been implicated in obesity-associated insulin resistance and type 2 diabetes. Yet, the contribution of inflammatory cells to the pathogenesis of skeletal muscle insulin resistance remains elusive. In a large cohort of obese human individuals, blood monocyte Fas (CD95) expression correlated with systemic and skeletal muscle insulin resistance. To test a causal role for myeloid cell Fas expression in the development of skeletal muscle insulin resistance, we generated myeloid/haematopoietic cell-specific Fas-depleted mice. Myeloid/haematopoietic Fas deficiency prevented the development of glucose intolerance in high fat-fed mice, in ob/ob mice, and in mice acutely challenged by LPS. In vivo, ex vivo and in vitro studies demonstrated preservation of muscle insulin responsiveness with no effect on adipose tissue or liver. Studies using neutralizing antibodies demonstrated a role for TNFα as mediator between myeloid Fas and skeletal muscle insulin resistance, supported by significant correlations between monocyte Fas expression and circulating TNFα in humans. In conclusion, our results demonstrate an unanticipated crosstalk between myeloid cells and skeletal muscle in the development of obesity-associated insulin resistance.
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http://dx.doi.org/10.1002/emmm.201302962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3936487PMC
January 2014

Engineering of a functional bone organ through endochondral ossification.

Proc Natl Acad Sci U S A 2013 Mar 11;110(10):3997-4002. Epub 2013 Feb 11.

Department of Surgery, University Hospital Basel, 4056 Basel, Switzerland.

Embryonic development, lengthening, and repair of most bones proceed by endochondral ossification, namely through formation of a cartilage intermediate. It was previously demonstrated that adult human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) can execute an endochondral program and ectopically generate mature bone. Here we hypothesized that hMSCs pushed through endochondral ossification can engineer a scaled-up ossicle with features of a "bone organ," including physiologically remodeled bone, mature vasculature, and a fully functional hematopoietic compartment. Engineered hypertrophic cartilage required IL-1β to be efficiently remodeled into bone and bone marrow upon subcutaneous implantation. This model allowed distinguishing, by analogy with bone development and repair, an outer, cortical-like perichondral bone, generated mainly by host cells and laid over a premineralized area, and an inner, trabecular-like, endochondral bone, generated mainly by the human cells and formed over the cartilaginous template. Hypertrophic cartilage remodeling was paralleled by ingrowth of blood vessels, displaying sinusoid-like structures and stabilized by pericytic cells. Marrow cavities of the ossicles contained phenotypically defined hematopoietic stem cells and progenitor cells at similar frequencies as native bones, and marrow from ossicles reconstituted multilineage long-term hematopoiesis in lethally irradiated mice. This study, by invoking a "developmental engineering" paradigm, reports the generation by appropriately instructed hMSC of an ectopic "bone organ" with a size, structure, and functionality comparable to native bones. The work thus provides a model useful for fundamental and translational studies of bone morphogenesis and regeneration, as well as for the controlled manipulation of hematopoietic stem cell niches in physiology and pathology.
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http://dx.doi.org/10.1073/pnas.1220108110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593845PMC
March 2013