Publications by authors named "Satoshi Yamazaki"

102 Publications

DHODH inhibition synergizes with DNA-demethylating agents in the treatment of myelodysplastic syndromes.

Blood Adv 2021 Jan;5(2):438-450

Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine nucleotide synthesis. DHODH inhibition has recently been recognized as a potential new approach for treating acute myeloid leukemia (AML) by inducing differentiation. We investigated the efficacy of PTC299, a novel DHODH inhibitor, for myelodysplastic syndrome (MDS). PTC299 inhibited the proliferation of MDS cell lines, and this was rescued by exogenous uridine, which bypasses de novo pyrimidine synthesis. In contrast to AML cells, PTC299 was inefficient at inhibiting growth and inducing the differentiation of MDS cells, but synergized with hypomethylating agents, such as decitabine, to inhibit the growth of MDS cells. This synergistic effect was confirmed in primary MDS samples. As a single agent, PTC299 prolonged the survival of mice in xenograft models using MDS cell lines, and was more potent in combination with decitabine. Mechanistically, a treatment with PTC299 induced intra-S-phase arrest followed by apoptotic cell death. Of interest, PTC299 enhanced the incorporation of decitabine, an analog of cytidine, into DNA by inhibiting pyrimidine production, thereby enhancing the cytotoxic effects of decitabine. RNA-seq data revealed the marked downregulation of MYC target gene sets with PTC299 exposure. Transfection of MDS cell lines with MYC largely attenuated the growth inhibitory effects of PTC299, suggesting MYC as one of the major targets of PTC299. Our results indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells and acts in a synergistic manner with decitabine. This combination therapy may be a new therapeutic option for the treatment of MDS.
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http://dx.doi.org/10.1182/bloodadvances.2020001461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839369PMC
January 2021

Limited rejuvenation of aged hematopoietic stem cells in young bone marrow niche.

J Exp Med 2021 Mar;218(3)

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.

Hematopoietic stem cells (HSCs) exhibit functional alterations, such as reduced regenerative capacity and myeloid-biased differentiation, with age. The HSC niche, which is essential for the maintenance of HSCs, also undergoes marked changes with aging. However, it has been technically challenging to directly evaluate the contribution of niche aging to age-associated HSC alterations without niche-damaging myeloablation in HSC transplantation assays. We herein transplanted an excess of aged HSCs into young mice without preconditioning. Although aged HSCs successfully engrafted in the intact young bone marrow niche, they poorly regenerated downstream progenitors and exhibited persistent myeloid-biased differentiation, resulting in no significant functional rejuvenation. Transcriptome and methylome analyses revealed that the young niche largely restored the transcriptional profile of aged HSCs, but not their DNA methylation profiles. Therefore, the restoration of the young niche is insufficient for rejuvenating HSC functions, highlighting a key role for age-associated cell-intrinsic defects in HSC aging.
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http://dx.doi.org/10.1084/jem.20192283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7690939PMC
March 2021

Efficacy of the novel tubulin polymerization inhibitor PTC-028 for myelodysplastic syndrome.

Cancer Sci 2020 Dec 2;111(12):4336-4347. Epub 2020 Nov 2.

Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Monomer tubulin polymerize into microtubules, which are highly dynamic and play a critical role in mitosis. Therefore, microtubule dynamics are an important target for anticancer drugs. The inhibition of tubulin polymerization or depolymerization was previously targeted and exhibited efficacy against solid tumors. The novel small molecule PTC596 directly binds tubulin, inhibits microtubule polymerization, downregulates MCL-1, and induces p53-independent apoptosis in acute myeloid leukemia cells. We herein investigated the efficacy of PTC-028, a structural analog of PTC596, for myelodysplastic syndrome (MDS). PTC-028 suppressed growth and induced apoptosis in MDS cell lines. The efficacy of PTC028 in primary MDS samples was confirmed using cell proliferation assays. PTC-028 synergized with hypomethylating agents, such as decitabine and azacitidine, to inhibit growth and induce apoptosis in MDS cells. Mechanistically, a treatment with PTC-028 induced G2/M arrest followed by apoptotic cell death. We also assessed the efficacy of PTC-028 in a xenograft mouse model of MDS using the MDS cell line, MDS-L, and the AkaBLI bioluminescence imaging system, which is composed of AkaLumine-HCl and Akaluc. PTC-028 prolonged the survival of mice in xenograft models. The present results suggest a chemotherapeutic strategy for MDS through the disruption of microtubule dynamics in combination with DNA hypomethylating agents.
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http://dx.doi.org/10.1111/cas.14684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734154PMC
December 2020

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16 Cells In Vivo.

Cell Metab 2020 Nov 18;32(5):814-828.e6. Epub 2020 Sep 18.

Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Electronic address:

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-Cre-tdTomato mouse model to analyze the in vivo characteristics of p16 cells at a single-cell level. We found tdTomato-positive p16 cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16 cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16 cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
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http://dx.doi.org/10.1016/j.cmet.2020.09.006DOI Listing
November 2020

Expert consensus regarding standardization of sample preparation for clotting time assays.

Int J Hematol 2020 Nov 14;112(5):614-620. Epub 2020 Sep 14.

Department of Laboratory Medicine, Kawasaki Medical School, Kurashiki, Japan.

Accurate clotting time assay results are vital, as the test is employed to indicate the amount of oral anticoagulant to be prescribed, while it is also used for screening the hemorrhagic and thrombotic diseases. The procedure chosen for preparation of a patient blood sample including centrifugation can contribute to significant differences in the results obtained. Thus, for the purpose of proposing a standardized method to appropriately prepare blood samples prior to assay, the Japanese Society of Laboratory Hematology organized the Working Group for Standardization of Sample Preparation for Clotting Time Assays (WG). Following reviews of previously announced guidelines and original experimental results, consensus was obtained by the WG, with the main findings as follows. (1) The recommended anticoagulant in the blood collection tube is sodium citrate solution at 0.105-0.109 M (3.13-3.2%). (2) Whole blood samples should be stored at room temperature (18-25 ˚C) within 1 h of collection from the patient. (3) For plasma preparation, centrifugation at 1500 × g should be performed for at least 15 min or at 2000 × g for at least 10 min at room temperature. (4) After the plasma sample is prepared, it should be stored at room temperature and assayed within 4 h.
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http://dx.doi.org/10.1007/s12185-020-02983-xDOI Listing
November 2020

The impact of cell maturation and tissue microenvironments on the expression of endosomal Toll-like receptors in monocytes and macrophages.

Int Immunol 2020 11;32(12):785-798

Division of Innate Immunity, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

Toll-like receptors (TLRs) impact myeloid cell responsiveness to environmental cues such as pathogen components and metabolites. Although TLR protein expression in monocytes and tissue macrophages is thought to be optimized for microenvironments in each tissue, a comprehensive study has not been reported. We here examined protein expression of endogenous TLRs in tissue-resident myeloid cells. Neutrophils in peripheral blood, spleen, liver and lung expressed TLR2, TLR4 and TLR5 in all tissues. Ly6C+ MHC II‒ classical monocytes mature into Ly6C‒ MHC II+ monocyte-derived dendritic cells (moDCs) or Ly6C‒ MHC II‒ patrolling monocytes. These subsets were found in all the tissues studied. TLR2 and TLR4 were displayed on all of these subsets, regardless of location. In contrast, expression of endosomal TLRs did vary with tissues and subsets. moDCs expressed TLR9, but much less TLR7. In contrast, TLR7, not TLR3 or TLR9, was highly expressed in classical and patrolling monocytes. Tissue macrophages such as red pulp macrophages in the spleen, Kupffer cells in the liver, microglia in the brain, alveolar macrophages in the lung and adipose tissue macrophages all expressed TLR2, TLR4 and TLR3. TLR7 was also expressed in these tissue macrophages except Kupffer cells in the liver. TLR9 expression in tissue macrophages was much lower or hard to detect. These results suggest that expression of endosomal TLRs in myeloid cells is influenced by their differentiation status and tissue-specific microenvironments.
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http://dx.doi.org/10.1093/intimm/dxaa055DOI Listing
November 2020

Activated HoxB4-induced hematopoietic stem cells from murine pluripotent stem cells via long-term programming.

Exp Hematol 2020 09 12;89:68-79.e7. Epub 2020 Aug 12.

Division of Molecular Therapy, Center for Experimental Medicine, The Institute of Medical Science, University of Tokyo, Tokyo, Japan. Electronic address:

Hematopoietic stem cells (HSCs) are multipotent cells that form the entire blood system and have the potential to cure several pathogenic conditions directly or indirectly arising from defects within the HSC compartment. Pluripotent stem cells (PSCs) or induced pluripotent stem cells (iPSCs) can give rise to all embryonic cell types; however, efficient in vitro differentiation of HSCs from PSCs remains challenging. HoxB4 is a key regulator orchestrating the differentiation of PSCs into all cells types across the mesodermal lineage, including HSCs. Moreover, the ectopic expression of HoxB4 enhances the in vitro generation and expansion of HSCs. However, several aspects of HoxB4 biology including its regulatory functions are not fully understood. Here, we describe the role of HoxB4 in indirectly inhibiting the emergence of mature CD45 HSCs from iPSCs in vitro. Forced activation of HoxB4 permitted long-term maintenance of functional hematopoietic stem and progenitor cells (HSPCs), which efficiently reconstituted hematopoiesis upon transplantation. Our method enables an easy and scalable in vitro platform for the generation of HSCs from iPSCs, which will ultimately lead to a better understanding of HSC biology and facilitate preparation of the roadma for producing an unrestricted supply of HSCs for several curative therapies.
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http://dx.doi.org/10.1016/j.exphem.2020.08.003DOI Listing
September 2020

Transcription factor 21 expression in injured podocytes of glomerular diseases.

Sci Rep 2020 07 13;10(1):11516. Epub 2020 Jul 13.

Department of Nephrology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan.

Transcription factor 21 (TCF21) is one of the essential transcription factors in kidney development. To elucidate its influence on glomerular disease, we have investigated TCF21 expression in human and rat kidney tissue, and its urinary concentration. Immunohistological analysis suggested the highest TCF21 expression in nephrotic syndrome along with the urinary protein level. Urinary TCF21 concentration in human showed a positive correlation with its podocyte expression level. In nephrotic rat models, TCF21 expression in podocytes increased along with the severity of nephrotic syndrome. Next, in vitro experiments using Tcf21-expressing murine podocyte cell line, we could observe some Tcf21-dependent effects, related with actin cytoskeleton dysregulation and apoptosis. Our study illustrated TCF21 expression changes in vivo and its in vitro-functional significance injured podocytes.
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http://dx.doi.org/10.1038/s41598-020-68422-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359327PMC
July 2020

HHEX promotes myeloid transformation in cooperation with mutant ASXL1.

Blood 2020 10;136(14):1670-1684

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

Additional sex combs-like 1 (ASXL1), an epigenetic modulator, is frequently mutated in myeloid neoplasms. Recent analyses of mutant ASXL1 conditional knockin (ASXL1-MT-KI) mice suggested that ASXL1-MT alone is insufficient for myeloid transformation. In our previous study, we used retrovirus-mediated insertional mutagenesis, which exhibited the susceptibility of ASXL1-MT-KI hematopoietic cells to transform into myeloid leukemia cells. In this screening, we identified the hematopoietically expressed homeobox (HHEX) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. Expression of HHEX enhanced proliferation of ASXL1-MT-expressing HSPCs by inhibiting apoptosis and blocking differentiation, whereas it showed only modest effect in normal HSPCs. Moreover, ASXL1-MT and HHEX accelerated the development of RUNX1-ETO9a and FLT3-ITD leukemia. Conversely, HHEX depletion profoundly attenuated the colony-forming activity and leukemogenicity of ASXL1-MT-expressing leukemia cells. Mechanistically, we identified MYB and ETV5 as downstream targets for ASXL1-MT and HHEX by using transcriptome and chromatin immunoprecipitation-next-generation sequencing analyses. Moreover, we found that expression of ASXL1-MT enhanced the binding of HHEX to the promoter loci of MYB or ETV5 via reducing H2AK119ub. Depletion of MYB or ETV5 induced apoptosis or differentiation in ASXL1-MT-expressing leukemia cells, respectively. In addition, ectopic expression of MYB or ETV5 reversed the reduced colony-forming activity of HHEX-depleted ASXL1-MT-expressing leukemia cells. These findings indicate that the HHEX-MYB/ETV5 axis promotes myeloid transformation in ASXL1-mutated preleukemia cells.
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http://dx.doi.org/10.1182/blood.2019004613DOI Listing
October 2020

A Sudden Onset of Severe Thrombocytopenia While Using Evolocumab.

Case Rep Hematol 2020 26;2020:3281626. Epub 2020 Mar 26.

Department of Endocrinology and Diabetes, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Iruma, Saitama 350-0495, Japan.

A 72-year-old man with a 10-year history of coronary heart disease started evolocumab treatment once a month after developing excess myalgia due to therapy with a 3-hydroxy-methylglutaryl CoA reductase inhibitor. No side effects such as myalgia symptoms had been reported during the first 14 months of evolocumab treatment; however, he suddenly presented with acute severe thrombocytopenia following the 14th treatment. His platelet count continued to decrease to a nadir of 1,000/L. His platelet-associated immunoglobulin G level had elevated to 790 ng/10 cells. He started receiving a combination of steroid therapy, high-dose immunoglobulin therapy, and platelet transfusions, but the first-line therapy was ineffective. He was subsequently treated with a thrombopoietin receptor agonist, and his platelet count recovered to 250,000/L.
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http://dx.doi.org/10.1155/2020/3281626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136763PMC
March 2020

In vivo expansion of functional human hematopoietic stem progenitor cells by butyzamide.

Int J Hematol 2020 May 12;111(5):739-741. Epub 2020 Mar 12.

Division of Stem Cell Biology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.

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http://dx.doi.org/10.1007/s12185-020-02849-2DOI Listing
May 2020

Long-term ex vivo expansion of mouse hematopoietic stem cells.

Nat Protoc 2020 02 8;15(2):628-648. Epub 2020 Jan 8.

Division of Stem Cell Biology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Utilizing multipotent and self-renewing capabilities, hematopoietic stem cells (HSCs) can maintain hematopoiesis throughout life. However, the mechanism behind such remarkable abilities remains undiscovered, at least in part because of the paucity of HSCs and the modest ex vivo expansion of HSCs in media that contain poorly defined albumin supplements such as bovine serum albumin. Here, we describe a simple platform for the expansion of functional mouse HSCs ex vivo for >1 month under fully defined albumin-free conditions. The culture system affords 236- to 899-fold expansion over the course of a month and is also amenable to clonal analysis of HSC heterogeneity. The large numbers of expanded HSCs enable HSC transplantation into nonconditioned recipients, which is otherwise not routinely feasible because of the large numbers of HSCs required. This protocol therefore provides a powerful approach with which to interrogate HSC self-renewal and lineage commitment and, more broadly, to study and characterize the hematopoietic and immune systems.
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http://dx.doi.org/10.1038/s41596-019-0263-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206416PMC
February 2020

Efficacy of a mitochondrion-targeting agent for reducing the level of urinary protein in rats with puromycin aminonucleoside-induced minimal-change nephrotic syndrome.

PLoS One 2020 6;15(1):e0227414. Epub 2020 Jan 6.

Department of Pediatrics, Osaka Medical College, Takatsuki, Osaka, Japan.

Background: Oxidative stress is a major factor responsible for minimal-change nephrotic syndrome (MCNS), which occurs most commonly in children. However, the influence of oxidative stress localized to mitochondria remains unclear. We examined the effect of a mitochondrion-targeting antioxidant, MitoTEMPO, in rats with puromycin aminonucleoside (PAN)-induced MCNS to clarify the degree to which mitochondrial oxidative stress affects MCNS.

Materials And Methods: Thirty Wistar rats were divided into three groups: normal saline group (n = 7), PAN group (n = 12), and PAN + MitoTEMPO group (n = 11). Rats in the PAN and PAN + MitoTEMPO groups received PAN on day 1, and those in the PAN + MitoTEMPO group received MitoTEMPO on days 0 to 9. Whole-day urine samples were collected on days 3 and 9, and samples of glomeruli and blood were taken for measurement of lipid peroxidation products. We also estimated the mitochondrial damage score in podocytes in all 3 groups using electron microscopy.

Results: Urinary protein excretion on day 9 and the levels of lipid peroxidation products in urine, glomeruli, and blood were significantly lower in the PAN + MitoTEMPO group than in the PAN group (p = 0.0019, p = 0.011, p = 0.039, p = 0.030). The mitochondrial damage score in podocytes was significantly lower in the PAN + MitoTEMPO group than in the PAN group (p <0.0001).

Conclusions: This mitochondrion-targeting agent was shown to reduce oxidative stress and mitochondrial damage in a MCNS model. A radical scavenger targeting mitochondria could be a promising drug for treatment of MCNS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227414PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944386PMC
April 2020

Use of polyvinyl alcohol for chimeric antigen receptor T-cell expansion.

Exp Hematol 2019 12 23;80:16-20. Epub 2019 Dec 23.

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA; Department of Genetics, Stanford University School of Medicine, Stanford, CA. Electronic address:

Serum albumin has long been an essential supplement for ex vivo hematopoietic and immune cell cultures. However, serum albumin medium supplements represent a major source of biological contamination in cell cultures and often cause loss of cellular function. As serum albumin exhibits significant batch-to-batch variability, it has also been blamed for causing major issues in experimental reproducibility. We recently discovered the synthetic polymer polyvinyl alcohol (PVA) as an inexpensive, Good Manufacturing Practice-compatible, and biologically inert serum albumin replacement for ex vivo hematopoietic stem cell cultures. Importantly, PVA is free of the biological contaminants that have plagued serum albumin-based media. Here, we describe that PVA can replace serum albumin in a range of blood and immune cell cultures including cell lines, primary leukemia samples, and human T lymphocytes. PVA can even replace human serum in the generation and expansion of functional chimeric antigen receptor (CAR) T cells, offering a potentially safer and more cost-efficient approach for this clinical cell therapy. In summary, PVA represents a chemically defined, biologically inert, and inexpensive alternative to serum albumin for a range of cell cultures in hematology and immunology.
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http://dx.doi.org/10.1016/j.exphem.2019.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7194120PMC
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

Akt inhibition synergizes with polycomb repressive complex 2 inhibition in the treatment of multiple myeloma.

Cancer Sci 2019 Dec 22;110(12):3695-3707. Epub 2019 Oct 22.

Department of Transfusion Medicine and Cell Therapy, Chiba University Hospital, Chiba, Japan.

Polycomb repressive complex 2 (PRC2) components, EZH2 and its homolog EZH1, and PI3K/Akt signaling pathway are focal points as therapeutic targets for multiple myeloma. However, the exact crosstalk between their downstream targets remains unclear. We herein elucidated some epigenetic interactions following Akt inhibition and demonstrated the efficacy of the combined inhibition of Akt and PRC2. We found that TAS-117, a potent and selective Akt inhibitor, downregulated EZH2 expression at the mRNA and protein levels via interference with the Rb-E2F pathway, while EZH1 was compensatively upregulated to maintain H3K27me3 modifications. Consistent with these results, the dual EZH2/EZH1 inhibitor, UNC1999, but not the selective EZH2 inhibitor, GSK126, synergistically enhanced TAS-117-induced cytotoxicity and provoked myeloma cell apoptosis. RNA-seq analysis revealed the activation of the FOXO signaling pathway after TAS-117 treatment. FOXO3/4 mRNA and their downstream targets were upregulated with the enhanced nuclear localization of FOXO3 protein after TAS-117 treatment. ChIP assays confirmed the direct binding of FOXO3 to EZH1 promoter, which was enhanced by TAS-117 treatment. Moreover, FOXO3 knockdown repressed EZH1 expression. Collectively, the present results reveal some molecular interactions between Akt signaling and epigenetic modulators, which emphasize the benefits of targeting PRC2 full activity and the Akt pathway as a therapeutic option for multiple myeloma.
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http://dx.doi.org/10.1111/cas.14207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890440PMC
December 2019

Generation of a human induced pluripotent stem cell line, IMSUTi002-A-1, harboring the leukemia-specific fusion gene ETV6-RUNX1.

Stem Cell Res 2019 10 22;40:101546. Epub 2019 Aug 22.

The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Reprograming human primary cancer cells remains technically challenging, and leukemia-specific human induced pluripotent stem cell (hiPSC) lines have not yet been generated from tissues of patients with acute leukemia (Muñoz-López et al., 2016). Here, we developed an hiPSC line with a doxycycline-inducible expression system with the fusion gene ETV6-RUNX1 integrated into the genome. This is the most common genetic aberration found in childhood leukemia. This hiPSC line will be a useful model for research on childhood leukemia pathology and risk assessment.
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http://dx.doi.org/10.1016/j.scr.2019.101546DOI Listing
October 2019

Bmi1 restricts the adipogenic differentiation of bone marrow stromal cells to maintain the integrity of the hematopoietic stem cell niche.

Exp Hematol 2019 08 11;76:24-37. Epub 2019 Aug 11.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan; Division of Stem Cell and Molecular Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan. Electronic address:

The polycomb group protein Bmi1 maintains hematopoietic stem cell (HSC) functions. We previously reported that Bmi1-deficient mice exhibited progressive fatty changes in bone marrow (BM). A large portion of HSCs reside in the perivascular niche created partly by endothelial cells and leptin receptor (LepR) BM stromal cells. To clarify how Bmi1 regulates the HSC niche, we specifically deleted Bmi1 in LepR cells in mice. The Bmi1 deletion promoted the adipogenic differentiation of LepR stromal cells and caused progressive fatty changes in the BM of limb bones with age, resulting in reductions in the numbers of HSCs and progenitors in BM and enhanced extramedullary hematopoiesis. This adipogenic change was also evident during BM regeneration after irradiation. Several adipogenic regulator genes appeared to be regulated by Bmi1. Our results indicate that Bmi1 keeps the adipogenic differentiation program repressed in BM stromal cells to maintain the integrity of the HSC niche.
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http://dx.doi.org/10.1016/j.exphem.2019.07.006DOI Listing
August 2019

Long-term eradication of extranodal natural killer/T-cell lymphoma, nasal type, by induced pluripotent stem cell-derived Epstein-Barr virus-specific rejuvenated T cells .

Haematologica 2020 03 11;105(3):796-807. Epub 2019 Jul 11.

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

Functionally rejuvenated induced pluripotent stem cell (iPSC)-derived antigen-specific cytotoxic T lymphocytes (CTL) are expected to be a potent immunotherapy for tumors. When L-asparaginase-containing standard chemotherapy fails in extranodal natural killer/T-cell lymphoma, nasal type (ENKL), no effective salvage therapy exists. The clinical course then is miserable. We demonstrate prolonged and robust eradication of ENKL by Epstein-Barr virus-specific iPSC-derived antigen-specific CTL, with iPSC-derived antigen-specific CTL persisting as central memory T cells in the mouse spleen for at least six months. The anti-tumor response is so strong that any concomitant effect of the programmed cell death 1 (PD-1) blockade is unclear. These results suggest that long-term persistent Epstein-Barr virus-specific iPSC-derived antigen-specific CTL contribute to a continuous anti-tumor effect and offer an effective salvage therapy for relapsed and refractory ENKL.
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http://dx.doi.org/10.3324/haematol.2019.223511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049350PMC
March 2020

Author Correction: Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.

Nature 2019 Jul;571(7766):E12

Division of Stem Cell Therapy, Distinguished Professor Unit, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41586-019-1395-9DOI Listing
July 2019

Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.

Nature 2019 07 29;571(7763):117-121. Epub 2019 May 29.

Division of Stem Cell Therapy, Distinguished Professor Unit, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche, stable ex vivo HSC expansion has previously been unattainable. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1 month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50 cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.
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http://dx.doi.org/10.1038/s41586-019-1244-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7006049PMC
July 2019

Mesenchymal-Epithelial Interactome Analysis Reveals Essential Factors Required for Fibroblast-Free Alveolosphere Formation.

iScience 2019 Jan 26;11:318-333. Epub 2018 Dec 26.

Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan; Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute of Biomedical Sciences, Tokyo University of Science, Noda 278-0022, Japan. Electronic address:

Lung epithelial cells and fibroblasts are key cell populations in lung development. Fibroblasts support type 2 alveolar epithelial cells (AEC2) in the developing and mature lung. However, fibroblast-AEC2 interactions have not been clearly described. We addressed this in the present study by time course serial analysis of gene expression sequencing (SAGE-seq) of epithelial cells and fibroblasts of developing and mature murine lungs. We identified lung fibroblast-epithelial interactions that potentially regulate alveologenesis and are mediated by fibroblast-expressed ligands and epithelial cell surface receptors. In the epithelial-fibroblast co-culture alveolosphere formation assay, single intervention against fibroblast-expressed ligand or associated signaling cascades promoted or inhibited alveolosphere growth. Adding the ligand-associated molecules fibroblast growth factor 7 and Notch ligand and inhibitors of bone morphogenetic protein 4, transforming growth factor β, and glycogen synthase kinase-3β to the culture medium enabled fibroblast-free alveolosphere formation. The results revealed the essential factors regulating fibroblast-AEC2 interactions.
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http://dx.doi.org/10.1016/j.isci.2018.12.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6329323PMC
January 2019

Acetaminophen and Febrile Seizure Recurrences During the Same Fever Episode.

Pediatrics 2018 11 8;142(5). Epub 2018 Oct 8.

Department of Pediatrics, Osaka Medical College, Takatsuki, Osaka, Japan.

Objectives: To confirm the safety of using acetaminophen for febrile seizures (FSs) and to assess its efficacy in preventing FS recurrence during the same fever episode.

Methods: In this single-center, prospective, open, randomized controlled study, we included children and infants (age range: 6-60 months) with FSs who visited our hospital between May 1, 2015, and April 30, 2017. The effectiveness of acetaminophen was examined by comparing the recurrence rates of patients in whom rectal acetaminophen (10 mg/kg) was administered every 6 hours until 24 hours after the first convulsion (if the fever remained >38.0°C) to the rates of patients in whom no antipyretics were administered. No placebo was administered to controls. The primary outcome measure was FS recurrence during the same fever episode.

Results: We evaluated 423 patients; of these, 219 were in the rectal acetaminophen group, and 204 were in the no antipyretics group. In the univariate analysis, the FS recurrence rate was significantly lower in the rectal acetaminophen group (9.1%) than in the no antipyretics group (23.5%; < .001). Among the variables in the final multiple logistic regression analysis, rectal acetaminophen use was the largest contributor to the prevention of FS recurrence during the same fever episode (odds ratio: 5.6; 95% confidence interval: 2.3-13.3).

Conclusions: Acetaminophen is a safe antipyretic against FSs and has the potential to prevent FS recurrence during the same fever episode.
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http://dx.doi.org/10.1542/peds.2018-1009DOI Listing
November 2018

Generation of Vascular Endothelial Cells and Hematopoietic Cells by Blastocyst Complementation.

Stem Cell Reports 2018 10 20;11(4):988-997. Epub 2018 Sep 20.

Division of Stem Cell Therapy, Distinguished Professor Unit, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; Institute for Stem Cell Biology and Regenerative Medicine, Department of Genetics, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305, USA. Electronic address:

In the case of organ transplantation accompanied by vascular anastomosis, major histocompatibility complex mismatched vascular endothelial cells become a target for graft rejection. Production of a rejection-free, transplantable organ, therefore, requires simultaneous generation of vascular endothelial cells within the organ. To generate pluripotent stem cell (PSC)-derived vascular endothelial cells, we performed blastocyst complementation with a vascular endothelial growth factor receptor-2 homozygous mutant blastocyst. This mutation is embryonic lethal at embryonic (E) day 8.5-9.5 due to an early defect in endothelial and hematopoietic cells. The Flk-1 homozygous knockout chimeric mice survived to adulthood for over 1 year without any abnormality, and all vascular endothelial cells and hematopoietic cells were derived from the injected PSCs. This approach could be used in conjunction with other gene knockouts which induce organ deficiency to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are PSC derived.
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http://dx.doi.org/10.1016/j.stemcr.2018.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178562PMC
October 2018

Mutant ASXL1 cooperates with BAP1 to promote myeloid leukaemogenesis.

Nat Commun 2018 07 16;9(1):2733. Epub 2018 Jul 16.

Division of Cellular Therapy, Advanced Clinical Research Center, and Division of Stem Cell Signaling, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Tokyo, 1088639, Japan.

ASXL1 mutations occur frequently in myeloid neoplasms and are associated with poor prognosis. However, the mechanisms by which mutant ASXL1 induces leukaemogenesis remain unclear. In this study, we report mutually reinforcing effects between a C-terminally truncated form of mutant ASXL1 (ASXL1-MT) and BAP1 in promoting myeloid leukaemogenesis. BAP1 expression results in increased monoubiquitination of ASXL1-MT, which in turn increases the catalytic function of BAP1. This hyperactive ASXL1-MT/BAP1 complex promotes aberrant myeloid differentiation of haematopoietic progenitor cells and accelerates RUNX1-ETO-driven leukaemogenesis. Mechanistically, this complex induces upregulation of posterior HOXA genes and IRF8 through removal of H2AK119 ubiquitination. Importantly, BAP1 depletion inhibits posterior HOXA gene expression and leukaemogenicity of ASXL1-MT-expressing myeloid leukemia cells. Furthermore, BAP1 is also required for the growth of MLL-fusion leukemia cells with posterior HOXA gene dysregulation. These data indicate that BAP1, which has long been considered a tumor suppressor, in fact plays tumor-promoting roles in myeloid neoplasms.
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http://dx.doi.org/10.1038/s41467-018-05085-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048047PMC
July 2018

Hematopoietic insults damage bone marrow niche by activating p53 in vascular endothelial cells.

Exp Hematol 2018 07 27;63:41-51.e1. Epub 2018 Apr 27.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan; Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan. Electronic address:

Hematopoietic stem cells (HSCs) are exposed to various insults such as genotoxic stress, inflammation, and infection, which have a direct effect. These insults deplete, cause a functional decline in, and promote HSC aging and transformation. However, the impact of hematopoietic insults on niche cells remains largely unknown. We have reported previously that p53 is activated in blood vessels by various stresses, including hypoxia, inflammation, and aging, and contributes to tissue dysfunction and metabolic abnormalities. We hypothesized that hematopoietic insults also affect the bone marrow (BM) vascular niche. Here, we demonstrate that p53 becomes activated in BM endothelial cells upon hematopoietic stresses such as irradiation and chemotherapeutic treatments. The conditional activation of p53 in VE-cadherin vascular niche cells by deleting Mdm2 induces the expression of p53 target genes specifically in vascular endothelial cells, resulting in the dilation and collapse of vascular endothelial cells and reductions in perivascular mesenchymal stromal cell numbers. Consequently, hematopoietic stem cells (HSCs) fail to maintain dormancy, mobilize to the periphery, and are depleted significantly. Our results indicate that various hematopoietic insults affect HSCs, not only directly, but also indirectly by altering vascular integrity, which is critical for perivascular niche formation and maintenance of HSCs.
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http://dx.doi.org/10.1016/j.exphem.2018.04.006DOI Listing
July 2018

Spred1 Safeguards Hematopoietic Homeostasis against Diet-Induced Systemic Stress.

Cell Stem Cell 2018 05 26;22(5):713-725.e8. Epub 2018 Apr 26.

Division of Molecular Genetics, Cancer and Stem Cell Research Program, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan; WPI Nano Life Science Institute (WPI-Nano LSI), Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan. Electronic address:

Stem cell self-renewal is critical for tissue homeostasis, and its dysregulation can lead to organ failure or tumorigenesis. While obesity can induce varied abnormalities in bone marrow components, it is unclear how diet might affect hematopoietic stem cell (HSC) self-renewal. Here, we show that Spred1, a negative regulator of RAS-MAPK signaling, safeguards HSC homeostasis in animals fed a high-fat diet (HFD). Under steady-state conditions, Spred1 negatively regulates HSC self-renewal and fitness, in part through Rho kinase activity. Spred1 deficiency mitigates HSC failure induced by infection mimetics and prolongs HSC lifespan, but it does not initiate leukemogenesis due to compensatory upregulation of Spred2. In contrast, HFD induces ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in functional HSC failure, severe anemia, and myeloproliferative neoplasm-like disease. HFD-induced hematopoietic abnormalities are mediated partly through alterations to the gut microbiota. Together, these findings reveal that diet-induced stress disrupts fine-tuning of Spred1-mediated signals to govern HSC homeostasis.
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http://dx.doi.org/10.1016/j.stem.2018.04.002DOI Listing
May 2018

Branched-chain amino acid depletion conditions bone marrow for hematopoietic stem cell transplantation avoiding amino acid imbalance-associated toxicity.

Exp Hematol 2018 07 26;63:12-16.e1. Epub 2018 Apr 26.

Division of Stem Cell Therapy, Center for Stem Cell Biology and Regeneration Medicine, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. Electronic address:

Hematopoietic stem cells (HSCs) are used clinically in bone marrow (BM) transplantation due to their unique ability to reform the entire hematopoietic system. Recently, we reported that HSCs are highly sensitive to valine, one of the three branched-chain amino acids (BCAAs) in addition to isoleucine and leucine. Dietary depletion of valine could even be used as a conditioning regimen for HSC transplantation. Here, we report that HSCs are highly sensitive to the balance of BCAAs, with both proliferation and survival reduced by BCAA imbalance. However, low but balanced BCAA levels failed to rescue HSC maintenance. Importantly, in vivo depletion of all three BCAAs was significantly less toxic than depletion of valine only. We demonstrate that BCAA depletion can replace valine depletion as a safer alternative to BM conditioning. In summary, by determining HSC metabolic requirements, we can improve metabolic approaches to BM conditioning.
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http://dx.doi.org/10.1016/j.exphem.2018.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052250PMC
July 2018

The hematopoietic stem cell diet.

Int J Hematol 2018 Jun 31;107(6):634-641. Epub 2018 Mar 31.

Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo, 108-8639, Japan.

Hematopoietic stem cells (HSCs) are responsible for sustaining life-long blood formation or hematopoiesis and are also used clinically in a form of bone marrow transplantation, a curative cellular therapy for a range of hematological diseases. HSCs are maintained throughout adult life by a complex biological niche or microenvironment, which is thought to be composed of a range of cellular, molecular, and metabolic components. The metabolic components of the HSC niche have become of increasing interest over the past few years. It is now well-recognized that metabolic activity is intimately linked to HSC function, and dysregulation of these metabolic pathways result in hematological pathologies such as leukemia. Here, we review the recent progress in this field including our current understanding of the "dietary" requirements of HSCs and how nutrition influences HSC activity. These recent findings have suggested promising new metabolic approaches to improve clinical HSC transplantation and leukemia therapies.
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http://dx.doi.org/10.1007/s12185-018-2451-1DOI Listing
June 2018