Publications by authors named "Yuya Kunisaki"

45 Publications

A Germinal Center-Associated Microenvironmental Signature Reflects Malignant Phenotype and Outcome of DLBCL.

Blood Adv 2021 Oct 12. Epub 2021 Oct 12.

Kyushu University Hospital, Japan.

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy with varying prognosis after the gold standard rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Several prognostic models have been established by focusing primarily on characteristics of lymphoma cells themselves, including cell-of-origin, genomic alterations, and gene/protein expressions. However, the prognostic impact of the lymphoma microenvironment and its association with characteristics of lymphoma cells are not fully understood. Using the nCounter-based gene expression profiling of untreated DLBCL tissues, we here assess the clinical impact of lymphoma microenvironment on the clinical outcomes and pathophysiological, molecular signatures in DLBCL. The presence of normal germinal center (GC)-microenvironmental cells, including follicular T cells, macrophage/dendritic cells, and stromal cells, in lymphoma tissue indicates a positive therapeutic response. Our prognostic model, based on quantitation of transcripts from distinct GC-microenvironmental cell markers, clearly identified patients with graded prognosis independently of existing prognostic models. We observed increased incidences of genomic alterations and aberrant gene expression associated with poor prognosis in DLBCL tissues lacking GC-microenvironmental cells relative to those containing these cells. These data suggest that the loss of GC-associated microenvironmental signature dictates clinical outcomes of DLBCL patients reflecting the accumulation of "unfavorable" molecular signatures.
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http://dx.doi.org/10.1182/bloodadvances.2021004618DOI Listing
October 2021

Granulocyte collection by polymorphonuclear cell-targeting apheresis with medium-molecular-weight hydroxyethyl starch.

Int J Hematol 2021 Aug 28. Epub 2021 Aug 28.

Center for Cellular and Molecular Medicine, Kyushu University Hospital, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.

Granulocyte transfusion (GTX) is a therapeutic option for patients with prolonged neutropenia suffering from severe infections. Efficient granulocyte collection by apheresis from donors requires clear separation of granulocytes from red blood cells (RBCs), and infusion of high-molecular-weight (MW) hydroxyethyl starch (HES) facilitates RBC sedimentation. Recent research has shown that apheresis with medium-MW HES may prevent adverse effects of high-MW HES on donors, but the rationale for collection with medium-MW HES has yet to be evaluated. To validate the use of medium-MW HES, we first performed experiments with whole blood samples to determine how efficiently high-, medium- and low-MW HES separated granulocytes from RBCs, and found that medium-MW HES was just as efficient as high-MW HES. We also reviewed clinical data of granulocyte apheresis at our institution to evaluate granulocyte yields. Retrospective analysis of granulocyte collection revealed that apheresis with medium-MW HES yielded sufficient granulocytes for GTX and that donor anemia reduced collection efficiency. These results collectively may help us to establish a safer method for apheresis targeting polymorphonuclear granulocytes as an alternative to high-MW HES.
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http://dx.doi.org/10.1007/s12185-021-03207-6DOI Listing
August 2021

Optimization of lymphapheresis for manufacturing autologous CAR-T cells.

Int J Hematol 2021 Oct 17;114(4):449-458. Epub 2021 Jul 17.

Center for Cellular and Molecular Medicine, Kyushu University Hospital, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.

Collection of CD3+ lymphocytes via lymphapheresis is essential for manufacturing autologous chimeric antigen receptor (CAR) T cells. Optimization of timing and procedures for lymphapheresis for each patient is critical because patients often have progressive diseases and receive medications that could reduce T cell counts. We conducted a retrospective study of clinical data from 28 patients who underwent lymphapheresis for CD19-directed CAR-T therapy with tisagenlecleucel to identify factors that could affect CD3+ lymphocyte yields. The numbers of CD3+ cells in peripheral blood were significantly correlated with CD3+ cell yields (correlation coefficient r = 0.84), which enabled us to estimate the volume of blood to process before apheresis. We also found that small cell ratio (SCR) at the apheresis site precisely reflected the proportion of lymphocytes, especially in patients without circulating blasts (coefficient of determination: r = 0.9). We were able to predict the CD3+ cell yield and prevent excessive apheresis by measuring pre-apheresis circulating CD3+ cell counts and monitoring SCR. Collectively, these results will help us to establish a strategy for optimization of lymphapheresis procedures for CAR-T cell production on a patient-by-patient basis.
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http://dx.doi.org/10.1007/s12185-021-03191-xDOI Listing
October 2021

Platelet decrease and efficacy of platelet-rich plasma return following peripheral blood stem cell apheresis.

J Clin Apher 2021 Oct 16;36(5):687-696. Epub 2021 Jun 16.

Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medicine, Fukuoka, Japan.

Background: Peripheral blood stem cell (PBSC) transplantation is a key treatment option for hematological diseases and is widely performed in clinical practice. Platelet loss is one of the major complications of PBSC apheresis, and platelet-rich plasma (PRP) return is considered in case of platelet decrease following apheresis; however, little is known about the frequency and severity of platelet loss and the efficacy of PRP return postapheresis.

Methods: We assessed changes in platelet counts following PBSC-related apheresis in 270 allogeneic (allo)- and 105 autologous (auto)-PBSC settings. We also evaluated the efficacy of PRP transfusion on platelet recovery postapheresis.

Results: In both allo- and auto-PBSC settings, the preapheresis platelet count (range, 84-385 and 33-558 × 10 /L, respectively) decreased postapheresis (range, 57-292 and 20-429 × 10 /L, respectively), whereas severe platelet decrease (<50 × 10 /L) was only observed in auto-PBSC patients (n = 9). We confirmed that platelet count before apheresis was a risk factor for severe platelet decrease (<50 × 10 /L) following auto-PBSC apheresis (odds ratio 0.749, P < .049). PRP return postapheresis facilitated platelet recovery in more than 80% of cases in both allo and auto settings.

Conclusion: Lower platelet count preapheresis is a useful predictor of severe platelet decrease following auto-PBSC apheresis and PRP return is an effective process to facilitate platelet recovery postapheresis.
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http://dx.doi.org/10.1002/jca.21917DOI Listing
October 2021

Transfer learning efficiently maps bone marrow cell types from mouse to human using single-cell RNA sequencing.

Commun Biol 2020 12 4;3(1):736. Epub 2020 Dec 4.

Centre for Human Development, Stem Cells and Regeneration, Faculty of Medicine, University of Southampton, Southampton, SO17 1BJ, UK.

Biomedical research often involves conducting experiments on model organisms in the anticipation that the biology learnt will transfer to humans. Previous comparative studies of mouse and human tissues were limited by the use of bulk-cell material. Here we show that transfer learning-the branch of machine learning that concerns passing information from one domain to another-can be used to efficiently map bone marrow biology between species, using data obtained from single-cell RNA sequencing. We first trained a multiclass logistic regression model to recognize different cell types in mouse bone marrow achieving equivalent performance to more complex artificial neural networks. Furthermore, it was able to identify individual human bone marrow cells with 83% overall accuracy. However, some human cell types were not easily identified, indicating important differences in biology. When re-training the mouse classifier using data from human, less than 10 human cells of a given type were needed to accurately learn its representation. In some cases, human cell identities could be inferred directly from the mouse classifier via zero-shot learning. These results show how simple machine learning models can be used to reconstruct complex biology from limited data, with broad implications for biomedical research.
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http://dx.doi.org/10.1038/s42003-020-01463-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718277PMC
December 2020

Mitochondrial Protein Synthesis Is Essential for Terminal Differentiation of CD45 TER119Erythroid and Lymphoid Progenitors.

iScience 2020 Nov 7;23(11):101654. Epub 2020 Oct 7.

Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.

p32/C1qbp regulates mitochondrial protein synthesis and is essential for oxidative phosphorylation in mitochondria. Although dysfunction of p32/C1qbp impairs fetal development and immune responses, its role in hematopoietic differentiation remains unclear. Here, we found that mitochondrial dysfunction affected terminal differentiation of newly identified erythroid/B-lymphoid progenitors among CD45 Ter119 CD31 triple-negative cells (TNCs) in bone marrow. Hematopoietic cell-specific genetic deletion of p32/C1qbp (p32cKO) in mice caused anemia and B-lymphopenia without reduction of hematopoietic stem/progenitor cells. In addition, p32cKO mice were susceptible to hematopoietic stress with delayed recovery from anemia. p32/C1qbp-deficient CD51 TNCs exhibited impaired mitochondrial oxidation that consequently led to inactivation of mTORC1 signaling, which is essential for erythropoiesis. These findings uncover the importance of mitochondria, especially at the stage of TNCs during erythropoiesis, suggesting that dysregulation of mitochondrial protein synthesis is a cause of anemia and B-lymphopenia with an unknown pathology.
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http://dx.doi.org/10.1016/j.isci.2020.101654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578749PMC
November 2020

Pericytes in Bone Marrow.

Authors:
Yuya Kunisaki

Adv Exp Med Biol 2019 ;1122:101-114

Kyushu University Hospital, Center for Cellular and Molecular Medicine, Fukuoka, Japan.

Bone marrow environments are composed of multiple cell types, most of which are thought to be derived from mesenchymal stem cells. In mouse bone marrow, stromal cells with CD45 Tie2 CD90 CD51 CD105 phenotype, Nestin-GFP, CXCL12-abundant reticular (CAR) cells, PDGFRα Sca-1 or CD51 PDGFRα, and Prx-1-derived CD45 Ter119 PDGFRα Sca-1 populations select for MSC activity. There is evidence that these stromal cell populations display some significant overlap with each other and comprise important cellular constituents of the hematopoietic stem cell niche. Moreover, these mesenchymal cell populations share characteristics in their location as they all are found around bone marrow vessels (can be called "pericytes"). In this chapter, with reviewing the recent literatures, how the pericytes relate to physiological and pathological hematopoiesis is argued.
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http://dx.doi.org/10.1007/978-3-030-11093-2_6DOI Listing
August 2019

Regnase-1-mediated post-transcriptional regulation is essential for hematopoietic stem and progenitor cell homeostasis.

Nat Commun 2019 03 6;10(1):1072. Epub 2019 Mar 6.

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Osaka, Suita,, 565-0871, Japan.

The balance between self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) maintains hematopoietic homeostasis, failure of which can lead to hematopoietic disorder. HSPC fate is controlled by signals from the bone marrow niche resulting in alteration of the stem cell transcription network. Regnase-1, a member of the CCCH zinc finger protein family possessing RNAse activity, mediates post-transcriptional regulatory activity through degradation of target mRNAs. The precise function of Regnase-1 has been explored in inflammation-related cytokine expression but its function in hematopoiesis has not been elucidated. Here, we show that Regnase-1 regulates self-renewal of HSPCs through modulating the stability of Gata2 and Tal1 mRNA. In addition, we found that dysfunction of Regnase-1 leads to the rapid onset of abnormal hematopoiesis. Thus, our data reveal that Regnase-1-mediated post-transcriptional regulation is required for HSPC maintenance and suggest that it represents a leukemia tumor suppressor.
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http://dx.doi.org/10.1038/s41467-019-09028-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403248PMC
March 2019

Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth.

Sci Adv 2019 01 23;5(1):eaau9060. Epub 2019 Jan 23.

Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan.

Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type-dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2α-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.
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http://dx.doi.org/10.1126/sciadv.aau9060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357737PMC
January 2019

Dedifferentiation process driven by TGF-beta signaling enhances stem cell properties in human colorectal cancer.

Oncogene 2019 02 4;38(6):780-793. Epub 2018 Sep 4.

Department of Medicine and Biosystemic Sciences, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.

Cancer stem cells (CSCs) possess the capacity for self-renewal and the potential to differentiate into non-CSCs. The recent discoveries of dynamic equilibrium between CSCs and non-CSCs revealed the significance of acquiring CSC-like properties in non-CSCs as an important process in progression of cancer. The mechanism underlying acquisition of CSC-like properties has mainly been investigated in the context of epithelial-mesenchymal transition. Here, we demonstrate the dedifferentiation process may be an alternative mechanism in acquisition of CSC-like properties in human colorectal cancer cells. By exploring the single-cell gene expression analysis of organoids developed from CD44 CSCs, we identified TWIST1 as a key molecule for maintaining the undifferentiated state of cancer cells. Consistent with the finding, we found that TGF-beta signaling pathway, a regulator of TWIST1, was specifically activated in the undifferentiated CD44 CSCs in human colorectal cancer using microarray-based gene expression analysis and quantitative pathology imaging system. Furthermore, we showed that external stimulation with TGF-beta and the induction of TWIST1 converted CD44 non-CSCs into the undifferentiated CD44 CSCs, leading to the significant increment of CSCs in xenograft models. This study strongly suggests dedifferentiation driven by TGF-beta signaling enhances stem cell properties in human colorectal cancer.
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http://dx.doi.org/10.1038/s41388-018-0480-0DOI Listing
February 2019

Gastrointestinal Graft-versus-Host Disease Is a Risk Factor for Postengraftment Bloodstream Infection in Allogeneic Hematopoietic Stem Cell Transplant Recipients.

Biol Blood Marrow Transplant 2018 11 15;24(11):2302-2309. Epub 2018 Jun 15.

Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan. Electronic address:

Bloodstream infection (BSI) is a well-known cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients. Here, we conducted a retrospective study to assess the morbidity, etiology, risk factors, and outcomes of BSI in the postengraftment period (PE-BSI) after allo-HSCT. Forty-three of 316 patients (13.6%) developed 57 PE-BSI episodes, in which 62 pathogens were isolated: Gram-positive bacteria, gram-negative bacteria, and fungi, respectively, accounted for 54.8%, 35.5%, and 9.7% of the isolates. Multivariate analysis revealed methylprednisolone use for graft-versus-host disease (GVHD) prophylaxis (odds ratio [OR], 6.49; 95% confidence interval [CI], 1.49 to 28.2; P = .013) and acute gastrointestinal GVHD (GI-GVHD) (OR, 8.82; 95% CI, 3.99 to 19.5; P < .0001) as risk factors for developing PE-BSI. This finding suggested that GI-GVHD increases the risk of bacterial translocation and subsequent septicemia. Moreover, among patients with GI-GVHD, insufficient response to corticosteroids, presumably related to an intestinal dysbiosis, significantly correlated with this complication. Patients with PE-BSI presented worse outcome compared with those without (3-year overall survival, 47.0% versus 18.6%; P < .001). Close microbiologic monitoring for BSIs and minimizing intestinal dysbiosis may be crucial to break the vicious cycle between GI-GVHD and bacteremia and to improve transplant outcomes especially in patients who require additional immunosuppressants.
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http://dx.doi.org/10.1016/j.bbmt.2018.06.002DOI Listing
November 2018

Identification of unipotent megakaryocyte progenitors in human hematopoiesis.

Blood 2017 06 23;129(25):3332-3343. Epub 2017 Mar 23.

Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan; and.

The developmental pathway for human megakaryocytes remains unclear, and the definition of pure unipotent megakaryocyte progenitor is still controversial. Using single-cell transcriptome analysis, we have identified a cluster of cells within immature hematopoietic stem- and progenitor-cell populations that specifically expresses genes related to the megakaryocyte lineage. We used CD41 as a positive marker to identify these cells within the CD34CD38IL-3RαCD45RA common myeloid progenitor (CMP) population. These cells lacked erythroid and granulocyte-macrophage potential but exhibited robust differentiation into the megakaryocyte lineage at a high frequency, both in vivo and in vitro. The efficiency and expansion potential of these cells exceeded those of conventional bipotent megakaryocyte/erythrocyte progenitors. Accordingly, the CD41 CMP was defined as a unipotent megakaryocyte progenitor (MegP) that is likely to represent the major pathway for human megakaryopoiesis, independent of canonical megakaryocyte-erythroid lineage bifurcation. In the bone marrow of patients with essential thrombocythemia, the MegP population was significantly expanded in the context of a high burden of Janus kinase 2 mutations. Thus, the prospectively isolatable and functionally homogeneous human MegP will be useful for the elucidation of the mechanisms underlying normal and malignant human hematopoiesis.
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http://dx.doi.org/10.1182/blood-2016-09-741611DOI Listing
June 2017

Differential cytokine contributions of perivascular haematopoietic stem cell niches.

Nat Cell Biol 2017 03 20;19(3):214-223. Epub 2017 Feb 20.

Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

Arterioles and sinusoids of the bone marrow (BM) are accompanied by stromal cells that express nerve/glial antigen 2 (NG2) and leptin receptor (LepR), and constitute specialized niches that regulate quiescence and proliferation of haematopoietic stem cells (HSCs). However, how niche cells differentially regulate HSC functions remains unknown. Here, we show that the effects of cytokines regulating HSC functions are dependent on the producing cell sources. Deletion of chemokine C-X-C motif ligand 12 (Cxcl12) or stem cell factor (Scf) from all perivascular cells marked by nestin-GFP dramatically depleted BM HSCs. Selective Cxcl12 deletion from arteriolar NG2 cells, but not from sinusoidal LepR cells, caused HSC reductions and altered HSC localization in BM. By contrast, deletion of Scf in LepR cells, but not NG2 cells, led to reductions in BM HSC numbers. These results uncover distinct contributions of cytokines derived from perivascular cells in separate vascular niches to HSC maintenance.
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http://dx.doi.org/10.1038/ncb3475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5467892PMC
March 2017

Enhanced Reconstitution of Human Erythropoiesis and Thrombopoiesis in an Immunodeficient Mouse Model with Kit(Wv) Mutations.

Stem Cell Reports 2016 09 4;7(3):425-438. Epub 2016 Aug 4.

Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan; Center for Cellular and Molecular Medicine, Kyushu University Hospital, Fukuoka 812-8582, Japan. Electronic address:

In human-to-mouse xenograft models, reconstitution of human hematopoiesis is usually B-lymphoid dominant. Here we show that the introduction of homozygous Kit(Wv) mutations into C57BL/6.Rag2(null)Il2rg(null) mice with NOD-Sirpa (BRGS) strongly promoted human multi-lineage reconstitution. After xenotransplantation of human CD34(+)CD38(-) cord blood cells, these newly generated C57BL/6.Rag2(null)Il2rg(null)NOD-Sirpa Kit(Wv/Wv) (BRGSK(Wv/Wv)) mice showed significantly higher levels of human cell chimerism and long-term multi-lineage reconstitution compared with BRGS mice. Strikingly, this mouse displayed a robust reconstitution of human erythropoiesis and thrombopoiesis with terminal maturation in the bone marrow. Furthermore, depletion of host macrophages by clodronate administration resulted in the presence of human erythrocytes and platelets in the circulation. Thus, attenuation of mouse KIT signaling greatly enhances the multi-lineage differentiation of human hematopoietic stem and progenitor cells (HSPCs) in mouse bone marrow, presumably by outcompeting mouse HSPCs to occupy suitable microenvironments. The BRGSK(Wv/Wv) mouse model is a useful tool to study human multi-lineage hematopoiesis.
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http://dx.doi.org/10.1016/j.stemcr.2016.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031955PMC
September 2016

Fetal liver hematopoietic stem cell niches associate with portal vessels.

Science 2016 Jan 3;351(6269):176-80. Epub 2015 Dec 3.

Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research. Albert Einstein College of Medicine, Bronx, NY, USA. Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA. Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA.

Whereas the cellular basis of the hematopoietic stem cell (HSC) niche in the bone marrow has been characterized, the nature of the fetal liver niche is not yet elucidated. We show that Nestin(+)NG2(+) pericytes associate with portal vessels, forming a niche promoting HSC expansion. Nestin(+)NG2(+) cells and HSCs scale during development with the fractal branching patterns of portal vessels, tributaries of the umbilical vein. After closure of the umbilical inlet at birth, portal vessels undergo a transition from Neuropilin-1(+)Ephrin-B2(+) artery to EphB4(+) vein phenotype, associated with a loss of periportal Nestin(+)NG2(+) cells and emigration of HSCs away from portal vessels. These data support a model in which HSCs are titrated against a periportal vascular niche with a fractal-like organization enabled by placental circulation.
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http://dx.doi.org/10.1126/science.aad0084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706788PMC
January 2016

[The hematopoietic stem cell niche].

Authors:
Yuya Kunisaki

Rinsho Ketsueki 2015 Oct;56(10):1888-93

Kyushu University Hospital, Center for Molecular and Cellular Medicine.

Somatic stem cells self-renew to maintain tissue homeostasis for the lifetime of organisms through tightly controlled proliferation and differentiation. Hematopoietic stem cells (HSCs) are essentially required for hematopoietic homeostasis. Therefore, they not only ensure lifelong replenishment of all blood lineages, but also maintain a constant pool. Cell cycle quiescence is a critical feature contributing to stem cell maintenance. Recent studies have highlighted the importance of bone marrow (BM) microenvironments that regulate HSC functions (HSC niches). In the HSC field, there has been considerable interest and debate regarding whether or not quiescence and proliferation of HSCs is regulated by distinct niches. Previous reports suggest that quiescent HSCs reside near osteoblasts in the BM whereas actively cycling HSCs are found near sinusoids. However, this popular concept has not been supported by rigorous analyses. To gain more insight into the spatial localization of HSCs, we have developed a whole-mount staining technique that allows precise measurements of 3D distances of HSCs from structures and allows computational simulation to define the significance of these interactions. This novel approach has allowed us to uncover two distinct types of vessels associated with quiescent and proliferating HSCs and to underscore the importance of arteriolar vessels for stem cell quiescence. We will discuss the crosstalk between the two hematopoietic and mesenchymal stem cells with a review of the recent literature.
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http://dx.doi.org/10.11406/rinketsu.56.1888DOI Listing
October 2015

Neutrophil ageing is regulated by the microbiome.

Nature 2015 Sep 16;525(7570):528-32. Epub 2015 Sep 16.

Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

Blood polymorphonuclear neutrophils provide immune protection against pathogens, but may also promote tissue injury in inflammatory diseases. Although neutrophils are generally considered to be a relatively homogeneous population, evidence for heterogeneity is emerging. Under steady-state conditions, neutrophil heterogeneity may arise from ageing and replenishment by newly released neutrophils from the bone marrow. Aged neutrophils upregulate CXCR4, a receptor allowing their clearance in the bone marrow, with feedback inhibition of neutrophil production via the IL-17/G-CSF axis, and rhythmic modulation of the haematopoietic stem-cell niche. The aged subset also expresses low levels of L-selectin. Previous studies have suggested that in vitro-aged neutrophils exhibit impaired migration and reduced pro-inflammatory properties. Here, using in vivo ageing analyses in mice, we show that neutrophil pro-inflammatory activity correlates positively with their ageing whilst in circulation. Aged neutrophils represent an overly active subset exhibiting enhanced αMβ2 integrin activation and neutrophil extracellular trap formation under inflammatory conditions. Neutrophil ageing is driven by the microbiota via Toll-like receptor and myeloid differentiation factor 88-mediated signalling pathways. Depletion of the microbiota significantly reduces the number of circulating aged neutrophils and dramatically improves the pathogenesis and inflammation-related organ damage in models of sickle-cell disease or endotoxin-induced septic shock. These results identify a role for the microbiota in regulating a disease-promoting neutrophil subset.
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http://dx.doi.org/10.1038/nature15367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712631PMC
September 2015

[Distinct vascular niches determine hematopoietic stem cell fate].

Authors:
Yuya Kunisaki

Rinsho Ketsueki 2015 Jun;56(6):606-13

Kyushu University, Department of Medicine and Biosystemic Science / Center for Cellular and Molecular Medicine.

Identification of cellular constituents of the hematopoietic stem cell (HSC) niche has recently been the subject of intensive investigation. To investigate the spatial localization of the HSC niches in bone marrow, we have established a whole-mount immunofluorescence imaging technique in which the 3D spatial relationships between stromal structures and HSCs in the BM can be precisely determined. The imaging assessment combined with computational simulations has uncovered a significant association between HSCs and arterioles, ensheathed exclusively by rare Nestin(bright) NG2⁺ pericytes (Nes(peri) cells), distinct from sinusoid-associated Nestin(dim) Leptin receptor (LepR)⁺ (Nes(retic)) cells which reportedly represent peri-vascular niche cells. Depletion of NG2⁺ cells using NG2-creERTM / inducible diphtheria toxin receptor (iDTR) mice changed HSC localization away from arterioles, induced HSC cycling and reduced long-term repopulation of HSCs in BM, suggesting that periarteriolar NG2⁺ cells form quiescent niches for HSCs. These results form the basis of studies that will allow us to genetically dissect the functions of distinct vascular niches. This vascular niche model, in which arterioles and sinusoids differentially regulate HSC quiescence and proliferation, respectively, have implications for the behavior of healthy HSC and may be useful in the future for evaluating the niches for cancer (leukemic) stem cells.
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http://dx.doi.org/10.11406/rinketsu.56.606DOI Listing
June 2015

[Cancer stem cells and the niches].

Authors:
Yuya Kunisaki

Nihon Rinsho 2015 May;73(5):739-44

The fate of stem cells is tightly controlled by specialized microenvironments (niches). Cell cycle quiescence is a key behavior of stem cells, which protects them from being exhausted by exogenous insults. Since the discovery of cancer stem cells, which are quiescent and thus resistant to anti-cancer therapy, there has been considerable interest regarding whether or not there are distinct niches for quiescent and expanding cancer cells, respectively. In our recent study using whole-mount immunofluorescence imaging techniques, we found that arteriolar niches promote hematopoietic stem cell (HSC) dormancy and that the NG2+ peri-arteriolar niche cells themselves are quiescent, suggesting that bone marrow arterioles comprise a specialized microenvironment that promotes quiescence of both HSCs and niche cells. In this review, we will argue about the advance of our knowledge on normal stem cell niches and the roles of microenvironments in cancer.
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May 2015

Megakaryocytes regulate hematopoietic stem cell quiescence through CXCL4 secretion.

Nat Med 2014 Nov 19;20(11):1315-20. Epub 2014 Oct 19.

1] Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, New York, USA. [2] Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, USA. [3] Mount Sinai School of Medicine, New York, New York, USA. [4] Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA.

In the bone marrow, hematopoietic stem cells (HSCs) lodge in specialized microenvironments that tightly control the proliferative state of HSCs to adapt to the varying needs for replenishment of blood cells while also preventing HSC exhaustion. All putative niche cells suggested thus far have a nonhematopoietic origin. Thus, it remains unclear how feedback from mature cells is conveyed to HSCs to adjust their proliferation. Here we show that megakaryocytes (MKs) can directly regulate HSC pool size in mice. Three-dimensional whole-mount imaging revealed that endogenous HSCs are frequently located adjacent to MKs in a nonrandom fashion. Selective in vivo depletion of MKs resulted in specific loss of HSC quiescence and led to a marked expansion of functional HSCs. Gene expression analyses revealed that MKs are the source of chemokine C-X-C motif ligand 4 (CXCL4, also named platelet factor 4 or PF4) in the bone marrow, and we found that CXCL4 regulates HSC cell cycle activity. CXCL4 injection into mice resulted in a reduced number of HSCs because of their increased quiescence. By contrast, Cxcl4(-/-) mice exhibited an increased number of HSCs and increased HSC proliferation. Combined use of whole-mount imaging and computational modeling was highly suggestive of a megakaryocytic niche capable of independently influencing HSC maintenance by regulating quiescence. These results indicate that a terminally differentiated cell type derived from HSCs contributes to the HSC niche, directly regulating HSC behavior.
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http://dx.doi.org/10.1038/nm.3707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258871PMC
November 2014

Acute myelogenous leukemia-induced sympathetic neuropathy promotes malignancy in an altered hematopoietic stem cell niche.

Cell Stem Cell 2014 Sep 10;15(3):365-375. Epub 2014 Jul 10.

Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Electronic address:

Perivascular mesenchymal stem and progenitor cells (MSPCs) are critical for forming a healthy hematopoietic stem cell (HSC) niche. However, the interactions and influence of acute myelogenous leukemia (AML) stem cells with the microenvironment remain largely unexplored. We have unexpectedly found that neuropathy of the sympathetic nervous system (SNS) promotes leukemic bone marrow infiltration in an MLL-AF9 AML model. Development of AML disrupts SNS nerves and the quiescence of Nestin(+) niche cells, leading to an expansion of phenotypic MSPCs primed for osteoblastic differentiation at the expense of HSC-maintaining NG2(+) periarteriolar niche cells. Adrenergic signaling promoting leukemogenesis is transduced by the β2, but not β3, adrenergic receptor expressed on stromal cells of leukemic bone marrow. These results indicate that sympathetic neuropathy may represent a mechanism for the malignancy in order to co-opt the microenvironment and suggest separate mesenchymal niche activities for malignant and healthy hematopoietic stem cells in the bone marrow.
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http://dx.doi.org/10.1016/j.stem.2014.06.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156919PMC
September 2014

Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development.

Dev Cell 2014 May;29(3):340-9

Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Electronic address:

Mesenchymal stem and progenitor cells (MSPCs) contribute to bone marrow (BM) homeostasis by generating multiple types of stromal cells. MSPCs can be labeled in the adult BM by Nestin-GFP, whereas committed osteoblast progenitors are marked by Osterix expression. However, the developmental origin and hierarchical relationship of stromal cells remain largely unknown. Here, by using a lineage-tracing system, we describe three distinct waves of contributions of Osterix(+) cells in the BM. First, Osterix(+) progenitors in the fetal BM contribute to nascent bone tissues and transient stromal cells that are replaced in the adult marrow. Second, Osterix-expressing cells perinatally contribute to osteolineages and long-lived BM stroma, which have characteristics of Nestin-GFP(+) MSPCs. Third, Osterix labeling in the adult marrow is osteolineage-restricted, devoid of stromal contribution. These results uncover a broad expression profile of Osterix and raise the intriguing possibility that distinct waves of stromal cells, primitive and definitive, may organize the developing BM.
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http://dx.doi.org/10.1016/j.devcel.2014.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4051418PMC
May 2014

Influences of vascular niches on hematopoietic stem cell fate.

Int J Hematol 2014 Jun 23;99(6):699-705. Epub 2014 Apr 23.

Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Bronx, NY, 10461, USA,

The fate decision of hematopoietic stem cells (HSCs), quiescence, proliferation or differentiation, is uniquely determined by functionally specialized microenvironments defined as the HSC niches. However, whether quiescence and proliferation of HSCs are regulated by spatially distinct niches is unclear. Although various candidate stromal cells have been identified as potential niche cells, the spatial localization of quiescent HSCs in the bone marrow remains controversial. In our recent study, we have established whole-mount confocal immunofluorescence techniques, which allow us to precisely assess the localization of HSCs and their relationships with stromal structures. Furthermore, we have assessed the significance of these associations using a computational simulation. These novel analyses have revealed that quiescent HSCs are specifically associated with small caliber arterioles, which are predominantly distributed in the endosteal bone marrow while the associations with sinusoidal vessels or osteoblasts are not significant. Physical ablation of the arteriolar niche causes the shift of HSC localization to sinusoidal niches, where HSCs are switched into non-quiescent status. This new imaging analyses together with previous studies suggest the presence of spatially distinct vascular niches for quiescent and non-quiescent (proliferating) HSCs in the bone marrow.
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http://dx.doi.org/10.1007/s12185-014-1580-4DOI Listing
June 2014

Arteriolar niches maintain haematopoietic stem cell quiescence.

Nature 2013 Oct 9;502(7473):637-43. Epub 2013 Oct 9.

1] Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, New York 10461, USA [2] Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

Cell cycle quiescence is a critical feature contributing to haematopoietic stem cell (HSC) maintenance. Although various candidate stromal cells have been identified as potential HSC niches, the spatial localization of quiescent HSCs in the bone marrow remains unclear. Here, using a novel approach that combines whole-mount confocal immunofluorescence imaging techniques and computational modelling to analyse significant three-dimensional associations in the mouse bone marrow among vascular structures, stromal cells and HSCs, we show that quiescent HSCs associate specifically with small arterioles that are preferentially found in endosteal bone marrow. These arterioles are ensheathed exclusively by rare NG2 (also known as CSPG4)(+) pericytes, distinct from sinusoid-associated leptin receptor (LEPR)(+) cells. Pharmacological or genetic activation of the HSC cell cycle alters the distribution of HSCs from NG2(+) periarteriolar niches to LEPR(+) perisinusoidal niches. Conditional depletion of NG2(+) cells induces HSC cycling and reduces functional long-term repopulating HSCs in the bone marrow. These results thus indicate that arteriolar niches are indispensable for maintaining HSC quiescence.
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http://dx.doi.org/10.1038/nature12612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3821873PMC
October 2013

PDGFRα and CD51 mark human nestin+ sphere-forming mesenchymal stem cells capable of hematopoietic progenitor cell expansion.

J Exp Med 2013 Jul 17;210(7):1351-67. Epub 2013 Jun 17.

Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

The intermediate filament protein Nestin labels populations of stem/progenitor cells, including self-renewing mesenchymal stem cells (MSCs), a major constituent of the hematopoietic stem cell (HSC) niche. However, the intracellular location of Nestin prevents its use for prospective live cell isolation. Hence it is important to find surface markers specific for Nestin⁺ cells. In this study, we show that the expression of PDGFRα and CD51 among CD45⁻ Ter119⁻ CD31⁻ mouse bone marrow (BM) stromal cells characterizes a large fraction of Nestin⁺ cells, containing most fibroblastic CFUs, mesenspheres, and self-renewal capacity after transplantation. The PDGFRα⁺ CD51 ⁺subset of Nestin⁺ cells is also enriched in major HSC maintenance genes, supporting the notion that niche activity co-segregates with MSC activity. Furthermore, we show that PDGFRα⁺ CD51⁺ cells in the human fetal BM represent a small subset of CD146⁺ cells expressing Nestin and enriched for MSC and HSC niche activities. Importantly, cultured human PDGFRα⁺ CD51⁺ nonadherent mesenspheres can significantly expand multipotent hematopoietic progenitors able to engraft immunodeficient mice. These results thus indicate that the HSC niche is conserved between the murine and human species and suggest that highly purified nonadherent cultures of niche cells may represent a useful novel technology to culture human hematopoietic stem and progenitor cells.
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http://dx.doi.org/10.1084/jem.20122252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3698522PMC
July 2013

Rhythmic modulation of the hematopoietic niche through neutrophil clearance.

Cell 2013 May;153(5):1025-35

Department of Epidemiology, Atherothrombosis and Imaging, Fundación Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid 28029, Spain.

Unique among leukocytes, neutrophils follow daily cycles of release from and migration back into the bone marrow, where they are eliminated. Because removal of dying cells generates homeostatic signals, we explored whether neutrophil elimination triggers circadian events in the steady state. Here, we report that the homeostatic clearance of neutrophils provides cues that modulate the physiology of the bone marrow. We identify a population of CD62L(LO) CXCR4(HI) neutrophils that have "aged" in the circulation and are eliminated at the end of the resting period in mice. Aged neutrophils infiltrate the bone marrow and promote reductions in the size and function of the hematopoietic niche. Modulation of the niche depends on macrophages and activation of cholesterol-sensing nuclear receptors and is essential for the rhythmic egress of hematopoietic progenitors into the circulation. Our results unveil a process that synchronizes immune and hematopoietic rhythms and expand the ascribed functions of neutrophils beyond inflammation. PAPERFLICK:
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http://dx.doi.org/10.1016/j.cell.2013.04.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128329PMC
May 2013

Chemotherapy-induced bone marrow nerve injury impairs hematopoietic regeneration.

Nat Med 2013 Jun 5;19(6):695-703. Epub 2013 May 5.

Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, New York, New York, USA.

Anticancer chemotherapy drugs challenge hematopoietic tissues to regenerate but commonly produce long-term sequelae. Chemotherapy-induced deficits in hematopoietic stem or stromal cell function have been described, but the mechanisms mediating hematopoietic dysfunction remain unclear. Administration of multiple cycles of cisplatin chemotherapy causes substantial sensory neuropathy. Here we demonstrate that chemotherapy-induced nerve injury in the bone marrow of mice is a crucial lesion impairing hematopoietic regeneration. Using pharmacological and genetic models, we show that the selective loss of adrenergic innervation in the bone marrow alters its regeneration after genotoxic insult. Sympathetic nerves in the marrow promote the survival of constituents of the stem cell niche that initiate recovery. Neuroprotection by deletion of Trp53 in sympathetic neurons or neuroregeneration by administration of 4-methylcatechol or glial-derived neurotrophic factor (GDNF) promotes hematopoietic recovery. These results demonstrate the potential benefit of adrenergic nerve protection for shielding hematopoietic niches from injury.
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http://dx.doi.org/10.1038/nm.3155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3964478PMC
June 2013

CD169⁺ macrophages provide a niche promoting erythropoiesis under homeostasis and stress.

Nat Med 2013 Apr 17;19(4):429-36. Epub 2013 Mar 17.

Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, New York, USA.

A role for macrophages in erythropoiesis was suggested several decades ago when erythroblastic islands in the bone marrow, composed of a central macrophage surrounded by developing erythroblasts, were described. However, the in vivo role of macrophages in erythropoiesis under homeostatic conditions or in disease remains unclear. We found that specific depletion of CD169(+) macrophages markedly reduced the number of erythroblasts in the bone marrow but did not result in overt anemia under homeostatic conditions, probably because of concomitant alterations in red blood cell clearance. However, CD169(+) macrophage depletion significantly impaired erythropoietic recovery from hemolytic anemia, acute blood loss and myeloablation. Furthermore, macrophage depletion normalized the erythroid compartment in a JAK2(V617F)-driven mouse model of polycythemia vera, suggesting that erythropoiesis in polycythemia vera remains under the control of macrophages in the bone marrow and splenic microenvironments. These results indicate that CD169(+) macrophages promote late erythroid maturation and that modulation of the macrophage compartment may be a new strategy to treat erythropoietic disorders.
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http://dx.doi.org/10.1038/nm.3057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983996PMC
April 2013

Circadian control of the immune system.

Nat Rev Immunol 2013 Mar 8;13(3):190-8. Epub 2013 Feb 8.

Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

Circadian rhythms, which have long been known to play crucial roles in physiology, are emerging as important regulators of specific immune functions. Circadian oscillations of immune mediators coincide with the activity of the immune system, possibly allowing the host to anticipate and handle microbial threats more efficiently. These oscillations may also help to promote tissue recovery and the clearance of potentially harmful cellular elements from the circulation. This Review summarizes the current knowledge of circadian rhythms in the immune system and provides an outlook on potential future implications.
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http://dx.doi.org/10.1038/nri3386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4090048PMC
March 2013

Adrenergic nerves govern circadian leukocyte recruitment to tissues.

Immunity 2012 Aug 2;37(2):290-301. Epub 2012 Aug 2.

Ruth L. and David S. Gottesman Institute for Stem Cell Biology and Regenerative Medicine, Albert Einstein College of Medicine, New York, NY 10461, USA.

The multistep sequence leading to leukocyte migration is thought to be locally regulated at the inflammatory site. Here, we show that broad systemic programs involving long-range signals from the sympathetic nervous system (SNS) delivered by adrenergic nerves regulate rhythmic recruitment of leukocytes in tissues. Constitutive leukocyte adhesion and migration in murine bone marrow (BM) and skeletal-muscle microvasculature fluctuated with circadian peak values at night. Migratory oscillations, altered by experimental jet lag, were implemented by perivascular SNS fibers acting on β-adrenoreceptors expressed on nonhematopoietic cells and leading to tissue-specific, differential circadian oscillations in the expression of endothelial cell adhesion molecules and chemokines. We showed that these rhythms have physiological consequences through alteration of hematopoietic cell recruitment and overall survival in models of septic shock, sickle cell vaso-occlusion, and BM transplantation. These data provide unique insights in the leukocyte adhesion cascade and the potential for time-based therapeutics for transplantation and inflammatory diseases.
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http://dx.doi.org/10.1016/j.immuni.2012.05.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428436PMC
August 2012
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