Publications by authors named "Motohiko Oshima"

58 Publications

CD271+CD51+PALLADIN- human mesenchymal stromal cells possess enhanced ossicle-forming potential.

Stem Cells Dev 2021 Apr 30. Epub 2021 Apr 30.

Kumamoto University, 13205, Kumamoto, Japan, 860-8555;

Human mesenchymal stem/stromal cells (hMSCs), when engrafted into immunodeficient mice can form ectopic bone organs with hematopoietic stem cell supportive functions. However, the ability to do so, through a cartilage intermediate appears limited to 30% of donor bone marrow samples. Here, we characterize the heterogeneous nature of hMSCs and their ability to efficiently form humanized ossicles observed in "good donors" to correlate with the frequency and functionality of chondrocyte progenitors. Flow cytometry of putative hMSC markers were enriched in the CD271+CD51+ stromal cell subset, which also possessed enhanced hMSC activity as assessed by single-cell CFU-F and undifferentiated mesensphere formation. Transcriptome analysis of CD271+ cells presented upregulation of chondro/osteogenesis-related genes and those related to HSC/niche maintenance factors such as CXCL12 and ANGIOPOIETIN 1. Among the candidate genes selected to enrich for subsets with greater chondrogenic ability, cells negative for the actin cross-linker, PALLADIN displayed the greatest CFU-F potential. Our study contributes to a better characterization of ossicle-forming hMSCs and their efficient isolation for the optimized engineering of human bone organs.
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http://dx.doi.org/10.1089/scd.2021.0021DOI Listing
April 2021

Fibroblasts from different body parts exhibit distinct phenotypes in adult progeria Werner syndrome.

Aging (Albany NY) 2021 02 24;13(4):4946-4961. Epub 2021 Feb 24.

Department of Endocrinology, Hematology and Gerontology, Chiba University Graduate School of Medicine, Chuo-Ku, Chiba 260-8670, Japan.

Werner syndrome (WS), also known as adult progeria, is characterized by accelerated aging symptoms from a young age. Patients with WS experience painful intractable skin ulcers with calcifications in their extremities, subcutaneous lipoatrophy, and sarcopenia. However, there is no significant abnormality in the trunk skin, where the subcutaneous fat relatively accumulates. The cause of such differences between the limbs and trunk is unknown. To investigate the underlying mechanism behind these phenomena, we established and analyzed dermal fibroblasts from the foot and trunk of two WS patients. As a result, WS foot-derived fibroblasts showed decreased proliferative potential compared to that from the trunk, which correlated with the telomere shortening. Transcriptome analysis showed increased expression of genes involved in osteogenesis in the foot fibroblasts, while adipogenic and chondrogenic genes were downregulated in comparison with the trunk. Consistent with these findings, the adipogenic and chondrogenic differentiation capacity was significantly decreased in the foot fibroblasts . On the other hand, the osteogenic potential was mutually maintained and comparable in the foot and trunk fibroblasts. These distinct phenotypes in the foot and trunk fibroblasts are consistent with the clinical symptoms of WS and may partially explain the underlying mechanism of this disease phenotype.
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http://dx.doi.org/10.18632/aging.202696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7950285PMC
February 2021

Distinct effects of chondroitin sulfate on hematopoietic cells and the stromal microenvironment in bone marrow hematopoiesis.

Exp Hematol 2021 Apr 12;96:52-62.e5. Epub 2021 Feb 12.

Department of Stem Cell Transplantation, Niigata University Medical and Dental Hospital, Niigata, Japan. Electronic address:

The bone marrow (BM) microenvironment, known as the BM niche, regulates hematopoiesis but is also affected by interactions with hematopoietic cells. Recent evidence indicates that extracellular matrix components are involved in these interactions. Chondroitin sulfate (CS), a glycosaminoglycan, is a major component of the extracellular matrix; however, it is not known whether CS has a physiological role in hematopoiesis. Here, we analyzed the functions of CS in hematopoietic and niche cells. CSGalNAcT1, which encodes CS N-acetylgalactosaminyltransferase-1 (T1), a key enzyme in CS biosynthesis, was highly expressed in hematopoietic stem and progenitor cells (HSPCs) and endothelial cells (ECs), but not in mesenchymal stromal cells (MSCs) in BM. In T1 knockout (T1KO) mice, a greater number of HSPCs existed compared with the wild-type (WT), but HSPCs from T1KO mice showed significantly impaired repopulation in WT recipient mice on serial transplantation. RNA sequence analysis revealed the activation of IFN-α/β signaling and endoplasmic reticulum stress in T1KO HSPCs. In contrast, the number of WT HSPCs repopulated in T1KO recipient mice was larger than that in WT recipient mice after serial transplantation, indicating that the T1KO niche supports repopulation of HSPCs better than the WT niche. There was no obvious difference in the distribution of vasculature and MSCs between WT and T1KO BM, suggesting that CS loss alters vascular niche functions without affecting its structure. Our results revealed distinct roles of CS in hematopoietic cells and BM niche, indicating that crosstalk between these components is important to maintain homeostasis in BM.
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http://dx.doi.org/10.1016/j.exphem.2021.02.003DOI Listing
April 2021

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

Blood Adv 2021 01;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

The combination of the tubulin binding small molecule PTC596 and proteasome inhibitors suppresses the growth of myeloma cells.

Sci Rep 2021 Jan 22;11(1):2074. Epub 2021 Jan 22.

Department of Hematology, Chiba University Hospital, Chiba, Japan.

The novel small molecule PTC596 inhibits microtubule polymerization and its clinical development has been initiated for some solid cancers. We herein investigated the preclinical efficacy of PTC596 alone and in combination with proteasome inhibitors in the treatment of multiple myeloma (MM). PTC596 inhibited the proliferation of MM cell lines as well as primary MM samples in vitro, and this was confirmed with MM cell lines in vivo. PTC596 synergized with bortezomib or carfilzomib to inhibit the growth of MM cells in vitro. The combination treatment of PTC596 with bortezomib exerted synergistic effects in a xenograft model of human MM cell lines in immunodeficient mice and exhibited acceptable tolerability. Mechanistically, treatment with PTC596 induced cell cycle arrest at G2/M phase followed by apoptotic cell death, associated with the inhibition of microtubule polymerization. RNA sequence analysis also revealed that PTC596 and the combination with bortezomib affected the cell cycle and apoptosis in MM cells. Importantly, endoplasmic reticulum stress induced by bortezomib was enhanced by PTC596, providing an underlying mechanism of action of the combination therapy. Our results indicate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options to improve outcomes in patients with MM.
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http://dx.doi.org/10.1038/s41598-021-81577-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822878PMC
January 2021

OGT Regulates Hematopoietic Stem Cell Maintenance via PINK1-Dependent Mitophagy.

Cell Rep 2021 Jan;34(1):108579

Department of Stem Cell and Immune Regulation, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan. Electronic address:

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a unique enzyme introducing O-GlcNAc moiety on target proteins, and it critically regulates various cellular processes in diverse cell types. However, its roles in hematopoietic stem and progenitor cells (HSPCs) remain elusive. Here, using Ogt conditional knockout mice, we show that OGT is essential for HSPCs. Ogt is highly expressed in HSPCs, and its disruption induces rapid loss of HSPCs with increased reactive oxygen species and apoptosis. In particular, Ogt-deficient hematopoietic stem cells (HSCs) lose quiescence, cannot be maintained in vivo, and become vulnerable to regenerative and competitive stress. Interestingly, Ogt-deficient HSCs accumulate defective mitochondria due to impaired mitophagy with decreased key mitophagy regulator, Pink1, through dysregulation of H3K4me3. Furthermore, overexpression of PINK1 restores mitophagy and the number of Ogt-deficient HSCs. Collectively, our results reveal that OGT critically regulates maintenance and stress response of HSCs by ensuring mitochondrial quality through PINK1-dependent mitophagy.
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http://dx.doi.org/10.1016/j.celrep.2020.108579DOI Listing
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

PRC2 insufficiency causes p53-dependent dyserythropoiesis in myelodysplastic syndrome.

Leukemia 2021 04 21;35(4):1156-1165. Epub 2020 Aug 21.

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

EZH1 and EZH2 are enzymatic components of polycomb repressive complex (PRC) 2, which catalyzes histone H3K27 tri-methylation (H3K27me3) to repress the transcription of PRC2 target genes. We previously reported that the hematopoietic cell-specific Ezh2 deletion (Ezh2) induced a myelodysplastic syndrome (MDS)-like disease in mice. We herein demonstrated that severe PRC2 insufficiency induced by the deletion of one allele Ezh1 in Ezh2-deficient mice (Ezh1Ezh2) caused advanced dyserythropoiesis accompanied by a differentiation block and enhanced apoptosis in erythroblasts. p53, which is activated by impaired ribosome biogenesis in del(5q) MDS, was specifically activated in erythroblasts, but not in hematopoietic stem or progenitor cells in Ezh1Ezh2 mice. Cdkn2a, a major PRC2 target encoding p19, which activates p53 by inhibiting MDM2 E3 ubiquitin ligase, was de-repressed in Ezh1Ezh2 erythroblasts. The deletion of Cdkn2a as well as p53 rescued dyserythropoiesis in Ezh1Ezh2 mice, indicating that PRC2 insufficiency caused p53-dependent dyserythropoiesis via the de-repression of Cdkn2a. Since PRC2 insufficiency is often involved in the pathogenesis of MDS, the present results suggest that p53-dependent dyserythropoiesis manifests in MDS in the setting of PRC2 insufficiency.
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http://dx.doi.org/10.1038/s41375-020-01023-1DOI Listing
April 2021

Overexpression of RUNX3 Represses RUNX1 to Drive Transformation of Myelodysplastic Syndrome.

Cancer Res 2020 06 27;80(12):2523-2536. Epub 2020 Apr 27.

Laboratory of Transcriptional Regulation in Leukemogenesis, International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto Japan.

RUNX3, a RUNX family transcription factor, regulates normal hematopoiesis and functions as a tumor suppressor in various tumors in humans and mice. However, emerging studies have documented increased expression of RUNX3 in hematopoietic stem/progenitor cells (HSPC) of a subset of patients with myelodysplastic syndrome (MDS) showing a worse outcome, suggesting an oncogenic function for RUNX3 in the pathogenesis of hematologic malignancies. To elucidate the oncogenic function of RUNX3 in the pathogenesis of MDS , we generated a -expressing, -deficient mouse model with the pancytopenia and dysplastic blood cells characteristic of MDS in patients. RUNX3-expressing cells markedly suppressed the expression levels of Runx1, a critical regulator of hemaotpoiesis in normal and malignant cells, as well as its target genes, which included crucial tumor suppressors such as and . RUNX3 bound these genes and remodeled their Runx1-binding regions in Tet2-deficient cells. Overexpression of RUNX3 inhibited the transcriptional function of Runx1 and compromised hematopoiesis to facilitate the development of MDS in the absence of Tet2, indicating that is an oncogene. Furthermore, overexpression of RUNX3 activated the transcription of Myc target genes and rendered cells sensitive to inhibition of Myc-Max heterodimerization. Collectively, these results reveal the mechanism by which RUNX3 overexpression exerts oncogenic effects on the cellular function of and transcriptional program in Tet2-deficient stem cells to drive the transformation of MDS. SIGNIFICANCE: This study defines the oncogenic effects of transcription factor RUNX3 in driving the transformation of myelodysplastic syndrome, highlighting RUNX3 as a potential target for therapeutic intervention.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-3167DOI Listing
June 2020

Novel myocardial markers GADD45G and NDUFS5 identified by RNA-sequencing predicts left ventricular reverse remodeling in advanced non-ischemic heart failure: a retrospective cohort study.

BMC Cardiovasc Disord 2020 03 5;20(1):116. Epub 2020 Mar 5.

Department of Cardiovascular Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.

Background: Left ventricular reverse remodeling (LVRR) has been detected in non-ischemic dilated cardiomyopathy (NIDCM) patients following optimal treatment. However, its prediction with only conventional modalities is often difficult. This study sought to examine whether RNA sequencing (RNA-seq) of myocardium tissue samples could predict LVRR in NIDCM.

Methods: A total of 17 advanced NIDCM patients with left ventricular ejection fraction (LVEF) below 30% who underwent cardiac biopsy from Left ventricle (LV) were prospectively recruited. They received optimal treatment and followed with echocardiogram every 6 months. Based on LVRR status after 12 months of treatment, patients were divided into the reverse remodeling (RR) or non-RR group. Tissue samples were analyzed by RNA-seq, and a functional analysis of differentially expressed genes was carried out.

Results: There were eight and nine patients in the RR and non-RR groups, respectively. No difference was found in age, sex, disease duration, LV end-diastolic diameter, and LVEF between the two groups. There were 155 genes that were differentially expressed between the two groups. Nicotinamide adenine dinucleotide ubiquinone oxidoreductase subunit (NDUF)S5 and Growth arrest and DNA-damage-inducible protein (GADD)45G, along with several genes related to the mitochondrial respiratory chain and ribosome, were significantly downregulated in the RR as compared to the non-RR group.

Conclusion: GADD45G and NDUFS5 are potential biomarkers for LVRR in patients with advanced NIDCM.
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http://dx.doi.org/10.1186/s12872-020-01396-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059273PMC
March 2020

Molecular pathogenesis of progression to myeloid leukemia from TET-insufficient status.

Blood Adv 2020 03;4(5):845-854

Department of Hematology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.

Loss-of-function mutations in ten-eleven translocation-2 (TET2) are recurrent events in acute myeloid leukemia (AML) as well as in preleukemic hematopoietic stem cells (HSCs) of age-related clonal hematopoiesis. TET3 mutations are infrequent in AML, but the level of TET3 expression in HSCs has been found to decline with age. We examined the impact of gradual decrease of TET function in AML development by generating mice with Tet deficiency at various degrees. Tet2f/f and Tet3f/f mice were crossed with mice expressing Mx1-Cre to generate Tet2f/wtTet3f/fMx-Cre+ (T2ΔT3), Tet2f/fTet3f/wtMx-Cre+ (ΔT2T3), and Tet2f/fTet3f/fMx-Cre+ (ΔT2ΔT3) mice. All ΔT2ΔT3 mice died of aggressive AML at a median survival of 10.7 weeks. By comparison, T2ΔT3 and ΔT2T3 mice developed AML at longer latencies, with a median survival of ∼27 weeks. Remarkably, all 9 T2ΔT3 and 8 ΔT2T3 mice with AML showed inactivation of the remaining nontargeted Tet2 or Tet3 allele, respectively, owing to exonic loss in either gene or stop-gain mutations in Tet3. Recurrent mutations other than Tet3 were not noted in any mice by whole-exome sequencing. Spontaneous inactivation of residual Tet2 or Tet3 alleles is a recurrent genetic event during the development of AML with Tet insufficiency.
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http://dx.doi.org/10.1182/bloodadvances.2019001324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065477PMC
March 2020

Bmi1 counteracts hematopoietic stem cell aging by repressing target genes and enforcing the stem cell gene signature.

Biochem Biophys Res Commun 2020 01 1;521(3):612-619. Epub 2019 Nov 1.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan; 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. Electronic address:

Polycomb-group proteins are critical regulators of stem cells. We previously demonstrated that Bmi1, a component of polycomb repressive complex 1, defines the regenerative capacity of hematopoietic stem cells (HSCs). Here, we attempted to ameliorate the age-related decline in HSC function by modulating Bmi1 expression. The forced expression of Bmi1 did not attenuate myeloid-biased differentiation of aged HSCs. However, single cell transplantation assays revealed that the sustained expression of Bmi1 augmented the multi-lineage repopulating capacity of aged HSCs. Chromatin immunoprecipitation-sequencing of Bmi1 combined with an RNA sequence analysis showed that the majority of Bmi1 direct target genes are developmental regulator genes marked with a bivalent histone domain. The sustained expression of Bmi1 strictly maintained the transcriptional repression of their target genes and enforced expression of HSC signature genes in aged HSCs. Therefore, the manipulation of Bmi1 expression is a potential approach against impairments in HSC function with aging.
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http://dx.doi.org/10.1016/j.bbrc.2019.10.153DOI Listing
January 2020

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

KDM2B in polycomb repressive complex 1.1 functions as a tumor suppressor in the initiation of T-cell leukemogenesis.

Blood Adv 2019 09;3(17):2537-2549

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

KDM2B together with RING1B, PCGF1, and BCOR or BCORL1 comprise polycomb repressive complex 1.1 (PRC1.1), a noncanonical PRC1 that catalyzes H2AK119ub1. It binds to nonmethylated CpG islands through its zinc finger-CxxC DNA binding domain and recruits the complex to target gene loci. Recent studies identified the loss of function mutations in the PRC1.1 gene, and in human T-cell acute lymphoblastic leukemia (T-ALL). We previously reported that insufficiency induces T-ALL in mice, supporting a tumor suppressor role for BCOR. However, the function of BCOR responsible for tumor suppression, either its corepressor function for BCL6 or that as a component of PRC1.1, remains unclear. We herein examined mice specifically lacking the zinc finger-CxxC domain of KDM2B in hematopoietic cells. Similar to -deficient mice, -deficient mice developed lethal T-ALL mostly in a NOTCH1-dependent manner. A chromatin immunoprecipitation sequence analysis of thymocytes revealed the binding of KDM2B at promoter regions, at which BCOR and EZH2 colocalized. KDM2B target genes markedly overlapped with those of NOTCH1 in human T-ALL cells, suggesting that noncanonical PRC1.1 antagonizes NOTCH1-mediated gene activation. KDM2B target genes were expressed at higher levels than the others and were marked with high levels of H2AK119ub1 and H3K4me3, but low levels of H3K27me3, suggesting that KDM2B target genes are transcriptionally active or primed for activation. These results indicate that PRC1.1 plays a key role in restricting excessive transcriptional activation by active NOTCH1, thereby acting as a tumor suppressor in the initiation of T-cell leukemogenesis.
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http://dx.doi.org/10.1182/bloodadvances.2018028522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737409PMC
September 2019

Author Correction: Lineage-specific RUNX2 super-enhancer activates MYC and promotes the development of blastic plasmacytoid dendritic cell neoplasm.

Nat Commun 2019 Aug 28;10(1):3943. Epub 2019 Aug 28.

Laboratory of Transcriptional Regulation in Leukemogenesis, International Research Center for Medical Sciences (IRCMS), Kumamoto University, 2-2-1 Honjo, Chuo Ward, Kumamoto, 860-0811, 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/s41467-019-11919-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713738PMC
August 2019

Aging of spermatogonial stem cells by Jnk-mediated glycolysis activation.

Proc Natl Acad Sci U S A 2019 08 29;116(33):16404-16409. Epub 2019 Jul 29.

Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, 606-8501 Kyoto, Japan;

Because spermatogonial stem cells (SSCs) are immortal by serial transplantation, SSC aging in intact testes is considered to be caused by a deteriorated microenvironment. Here, we report a cell-intrinsic mode of SSC aging by glycolysis activation. Using cultured SSCs, we found that aged SSCs proliferated more actively than young SSCs and showed enhanced glycolytic activity. Moreover, they remained euploid and exhibited stable androgenetic imprinting patterns with robust SSC activity despite having shortened telomeres. Aged SSCs showed increased expression, which was associated with decreased Polycomb complex 2 activity. Our results suggest that aberrant expression activated c- N-terminal kinase (JNK), which down-regulated mitochondria numbers by suppressing Down-regulation of probably decreased reactive oxygen species and enhanced glycolysis. Analyses of the -deficient aging mouse model and 2-y-old aged rats confirmed JNK hyperactivation and increased glycolysis. Therefore, not only microenvironment but also intrinsic activation of JNK-mediated glycolysis contributes to SSC aging.
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http://dx.doi.org/10.1073/pnas.1904980116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697785PMC
August 2019

Genome-Wide Mapping of Bivalent Histone Modifications in Hepatic Stem/Progenitor Cells.

Stem Cells Int 2019 1;2019:9789240. Epub 2019 Apr 1.

Department of Gastroenterology, Graduate School of Medicine, Chiba University, Chiba, Japan.

The "bivalent domain," a distinctive histone modification signature, is characterized by repressive trimethylation of histone H3 at lysine 27 (H3K27me3) and active trimethylation of histone H3 at lysine 4 (H3K4me3) marks. Maintenance and dynamic resolution of these histone marks play important roles in regulating differentiation processes in various stem cell systems. However, little is known regarding their roles in hepatic stem/progenitor cells. In the present study, we conducted the chromatin immunoprecipitation (ChIP) assay followed by high-throughput DNA sequencing (ChIP-seq) analyses in purified delta-like 1 protein (Dlk) hepatic stem/progenitor cells and successfully identified 562 genes exhibiting bivalent domains within 2 kb of the transcription start site. Gene ontology analysis revealed that these genes were enriched in developmental functions and differentiation processes. Microarray analyses indicated that many of these genes exhibited derepression after differentiation toward hepatocyte and cholangiocyte lineages. Among these, 72 genes, including and , were significantly upregulated after differentiation toward hepatocyte or cholangiocyte lineages. Knockdown of in Dlk cells suppressed colony propagation and resulted in increased numbers of albumin/cytokeratin 7 progenitor cells in colonies. These findings implicate that derepression of expression is required to induce normal differentiation processes. In conclusion, combined ChIP-seq and microarray analyses successfully identified bivalent genes. Functional analyses of these genes will help elucidate the epigenetic machinery underlying the terminal differentiation of hepatic stem/progenitor cells.
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http://dx.doi.org/10.1155/2019/9789240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466853PMC
April 2019

Lineage-specific RUNX2 super-enhancer activates MYC and promotes the development of blastic plasmacytoid dendritic cell neoplasm.

Nat Commun 2019 04 10;10(1):1653. Epub 2019 Apr 10.

Laboratory of Transcriptional Regulation in Leukemogenesis, International Research Center for Medical Sciences (IRCMS), Kumamoto University, 2-2-1 Honjo, Chuo Ward, Kumamoto, 860-0811, Japan.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive subtype of acute leukemia, the cell of origin of which is considered to be precursors of plasmacytoid dendritic cells (pDCs). Since translocation (6;8)(p21;q24) is a recurrent anomaly for BPDCN, we demonstrate that a pDC-specific super-enhancer of RUNX2 is associated with the MYC promoter due to t(6;8). RUNX2 ensures the expression of pDC-signature genes in leukemic cells, but also confers survival and proliferative properties in BPDCN cells. Furthermore, the pDC-specific RUNX2 super-enhancer is hijacked to activate MYC in addition to RUNX2 expression, thereby promoting the proliferation of BPDCN. We also demonstrate that the transduction of MYC and RUNX2 is sufficient to initiate the transformation of BPDCN in mice lacking Tet2 and Tp53, providing a model that accurately recapitulates the aggressive human disease and gives an insight into the molecular mechanisms underlying the pathogenesis of BPDCN.
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http://dx.doi.org/10.1038/s41467-019-09710-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458132PMC
April 2019

The chromatin-binding protein Phf6 restricts the self-renewal of hematopoietic stem cells.

Blood 2019 06 27;133(23):2495-2506. Epub 2019 Mar 27.

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

Recurrent inactivating mutations have been identified in the X-linked plant homeodomain finger protein 6 () gene, encoding a chromatin-binding transcriptional regulator protein, in various hematological malignancies. However, the role of PHF6 in normal hematopoiesis and its tumor-suppressor function remain largely unknown. We herein generated mice carrying a floxed allele and inactivated in hematopoietic cells at various developmental stages. The deletion in embryos augmented the capacity of hematopoietic stem cells (HSCs) to proliferate in cultures and reconstitute hematopoiesis in recipient mice. The deletion in neonates and adults revealed that cycling HSCs readily acquired an advantage in competitive repopulation upon the deletion, whereas dormant HSCs only did so after serial transplantations. -deficient HSCs maintained an enhanced repopulating capacity during serial transplantations; however, they did not induce any hematological malignancies. Mechanistically, Phf6 directly and indirectly activated downstream effectors in tumor necrosis factor α (TNFα) signaling. The deletion repressed the expression of a set of genes associated with TNFα signaling, thereby conferring resistance against the TNFα-mediated growth inhibition on HSCs. Collectively, these results not only define Phf6 as a novel negative regulator of HSC self-renewal, implicating inactivating mutations in the pathogenesis of hematological malignancies, but also indicate that a deficiency alone is not sufficient to induce hematopoietic transformation.
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http://dx.doi.org/10.1182/blood.2019000468DOI Listing
June 2019

Deregulated Polycomb functions in myeloproliferative neoplasms.

Int J Hematol 2019 Aug 31;110(2):170-178. Epub 2019 Jan 31.

Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.

Polycomb proteins function in the maintenance of gene silencing via post-translational modifications of histones and chromatin compaction. Genetic and biochemical studies have revealed that the repressive function of Polycomb repressive complexes (PRCs) in transcription is counteracted by the activating function of Trithorax-group complexes; this balance fine-tunes the expression of genes critical for development and tissue homeostasis. The function of PRCs is frequently dysregulated in various cancer cells due to altered expression or recurrent somatic mutations in PRC genes. The tumor suppressive functions of EZH2-containing PRC2 and a PRC2-related protein ASXL1 have been investigated extensively in the pathogenesis of hematological malignancies, including myeloproliferative neoplasms (MPN). BCOR, a component of non-canonical PRC1, suppresses various hematological malignancies including MPN. In this review, we focus on recent findings on the role of PRCs in the pathogenesis of MPN and the therapeutic impact of targeting the pathological functions of PRCs in MPN.
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http://dx.doi.org/10.1007/s12185-019-02600-6DOI Listing
August 2019

Genetic and transcriptional landscape of plasma cells in POEMS syndrome.

Leukemia 2019 07 11;33(7):1723-1735. Epub 2019 Jan 11.

Department of Hematology, Chiba University Hospital, Chiba, Japan.

POEMS syndrome is a rare paraneoplastic disease associated with monoclonal plasma cells; however, the pathogenic importance of plasma cells remains unclear. We performed comprehensive genetic analyses of plasma cells in 20 patients with POEMS syndrome. Whole exome sequencing was performed in 11 cases and found a total of 308 somatic mutations in 285 genes. Targeted sequencing was performed in all 20 cases and identified 20 mutations in 7 recurrently mutated genes, namely KLHL6, LTB, EHD1, EML4, HEPHL1, HIPK1, and PCDH10. None of the driver gene mutations frequently found in multiple myeloma (MM) such as NRAS, KRAS, BRAF, and TP53 was detected. Copy number analysis showed chromosomal abnormalities shared with monoclonal gammopathy of undetermined significance (MGUS), suggesting a partial overlap in the early development of MGUS and POEMS syndrome. RNA sequencing revealed a transcription profile specific to POEMS syndrome when compared with normal plasma cells, MGUS and MM. Unexpectedly, disease-specific VEGFA expression was not increased in POEMS syndrome. Our study illustrates that the genetic and transcriptional profiles of plasma cells in POEMS syndrome are distinct from MM and MGUS, indicating unique function of clonal plasma cells in its pathogenesis.
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http://dx.doi.org/10.1038/s41375-018-0348-xDOI Listing
July 2019

Ezh1 Targets Bivalent Genes to Maintain Self-Renewing Stem Cells in Ezh2-Insufficient Myelodysplastic Syndrome.

iScience 2018 Nov 15;9:161-174. Epub 2018 Oct 15.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 Japan; Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Electronic address:

Polycomb repressive complex (PRC) 2 represses transcription through histone H3K27 trimethylation (H3K27me3). We previously reported that the hematopoietic-cell-specific deletion of Ezh2, encoding a PRC2 enzyme, induced myelodysplastic syndrome (MDS) in mice, whereas the concurrent Ezh1 deletion depleted hematopoietic stem and progenitor cells (HSPCs). We herein demonstrated that mice with only one Ezh1 allele (Ezh1Ezh2) maintained HSPCs. A chromatin immunopreciptation sequence analysis revealed that residual PRC2 preferentially targeted genes with high levels of H3K27me3 and H2AK119 monoubiquitination (H2AK119ub1) in HSPCs (designated as Ezh1 core target genes), which were mostly developmental regulators, and maintained H3K27me3 levels in Ezh1Ezh2 HSPCs. Even upon the complete depletion of Ezh1 and Ezh2, H2AK119ub1 levels were largely retained, and only a minimal number of Ezh1 core targets were de-repressed. These results indicate that genes marked with high levels of H3K27me3 and H2AK119ub1 are the core targets of polycomb complexes in HSPCs as well as MDS stem cells.
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http://dx.doi.org/10.1016/j.isci.2018.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223231PMC
November 2018

insufficiency promotes initiation and progression of myelodysplastic syndrome.

Blood 2018 12 18;132(23):2470-2483. Epub 2018 Sep 18.

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

, encoding BCL-6 corepressor (BCOR), is X-linked and targeted by somatic mutations in various hematological malignancies including myelodysplastic syndrome (MDS). We previously reported that mice lacking exon 4 ( ) in the hematopoietic compartment developed NOTCH-dependent acute T-cell lymphoblastic leukemia (T-ALL). Here, we analyzed mice lacking exons 9 and 10 ( ), which express a carboxyl-terminal truncated BCOR that fails to interact with core effector components of polycomb repressive complex 1.1. mice developed lethal T-ALL in a similar manner to mice, whereas hematopoietic cells showed a growth advantage in the myeloid compartment that was further enhanced by the concurrent deletion of mice developed lethal MDS with progressive anemia and leukocytopenia, inefficient hematopoiesis, and the morphological dysplasia of blood cells. MDS cells reproduced MDS or evolved into lethal MDS/myeloproliferative neoplasms in secondary recipients. Transcriptional profiling revealed the derepression of myeloid regulator genes of the family and cluster genes in progenitor cells and the activation of p53 target genes specifically in MDS erythroblasts where massive apoptosis occurred. Our results reveal a tumor suppressor function of BCOR in myeloid malignancies and highlight the impact of insufficiency on the initiation and progression of MDS.
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http://dx.doi.org/10.1182/blood-2018-01-827964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450057PMC
December 2018

Forkhead box protein A1 confers resistance to transforming growth factor-β-induced apoptosis in breast cancer cells through inhibition of Smad3 nuclear translocation.

J Cell Biochem 2018 Sep 11. Epub 2018 Sep 11.

Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.

Transforming growth factor-β (TGF-β) induces apoptosis of normal epithelial cells, such as mammary epithelium. Although breast cancer progression associates with acquisition of resistance to TGF-β-induced apoptosis, the molecular mechanisms underlying this resistance are largely unknown. Here, we show that forkhead box protein A1 (FOXA1), which is known as a pioneer transcription factor, suppresses TGF-β-induced apoptosis of estrogen receptor-positive breast cancer cells. FOXA1 is found to inhibit nuclear translocation of Smad3, a key transcription factor downstream of TGF-β signaling, through suppression of the binding of Smad3 to the nuclear import receptor importin7. Furthermore, RNA sequencing analyses show that knockdown of FOXA1 upregulates Smad3-mediated proapoptotic gene expression. These results demonstrate that FOXA1 as a potent survival factor that suppresses TGF-β-induced apoptosis by inhibiting Smad3 signaling in estrogen receptor-positive breast cancer cells. Thus, we provide evidence for the first time that FOXA1 localizing to the cytoplasm negatively regulates Smad3-induced apoptosis in TGF-β-mediated signal transduction.
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http://dx.doi.org/10.1002/jcb.27551DOI Listing
September 2018

Ezh2 loss propagates hypermethylation at T cell differentiation-regulating genes to promote leukemic transformation.

J Clin Invest 2018 08 6;128(9):3872-3886. Epub 2018 Aug 6.

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

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is a new pathological entity with poor outcomes in T cell ALL (T-ALL) that is characterized by a high incidence of loss-of-function mutations in polycomb repressive complex 2 (PRC2) genes. We generated a mouse model of ETP-ALL by deleting Ezh2, one of the PRC2 genes, in p53-null hematopoietic cells. The loss of Ezh2 in p53-null hematopoietic cells impeded the differentiation of ETPs and eventually induced ETP-ALL-like disease in mice, indicating that PRC2 functions as a bona fide tumor suppressor in ETPs. A large portion of PRC2 target genes acquired DNA hypermethylation of their promoters following reductions in H3K27me3 levels upon the loss of Ezh2, which included pivotal T cell differentiation-regulating genes. The reactivation of a set of regulators by a DNA-demethylating agent, but not the transduction of single regulator genes, effectively induced the differentiation of ETP-ALL cells. Thus, PRC2 protects key T cell developmental regulators from DNA hypermethylation in order to keep them primed for activation upon subsequent differentiation phases, while its insufficiency predisposes ETPs to leukemic transformation. These results revealed a previously unrecognized epigenetic switch in response to PRC2 dysfunction and provide the basis for specific rational epigenetic therapy for ETP-ALL with PRC2 insufficiency.
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http://dx.doi.org/10.1172/JCI94645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6118644PMC
August 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

Hmga2 collaborates with V617F in the development of myeloproliferative neoplasms.

Blood Adv 2017 Jun 14;1(15):1001-1015. Epub 2017 Jun 14.

Department of Cardiovascular Medicine, and.

High-mobility group AT-hook 2 () is crucial for the self-renewal of fetal hematopoietic stem cells (HSCs) but is downregulated in adult HSCs via repression by and the polycomb-recessive complex 2 (PRC2) including EZH2. The messenger RNA (mRNA) level is often elevated in patients with myelofibrosis that exhibits an advanced myeloproliferative neoplasm (MPN) subtype, and deletion of promotes the progression of severe myelofibrosis in mice with upregulation of several oncogenes such as . However, the direct role of in the pathogenesis of MPNs remains unknown. To clarify the impact of on MPNs carrying the driver mutation, we generated Δ/ mice overexpressing Hmga2 due to deletion of the 3' untranslated region. Compared with mice, Δ/ mice exhibited more severe leukocytosis, anemia and splenomegaly, and shortened survival, whereas severity of myelofibrosis was comparable. Δ/ cells showed a greater repopulating ability that reproduced the severe MPN compared with cells in serial bone marrow transplants, indicating that Hmga2 promotes MPN progression at the HSC level. Hmga2 also enhanced apoptosis of erythroblasts that may worsen anemia. Relative to hematopoietic stem and progenitor cells (HSPCs), over 30% of genes upregulated in Δ/ HSPCs overlapped with those derepressed by loss in / HSPCs, suggesting that Hmga2 may facilitate upregulation of Ezh2 targets. Correspondingly, deletion of ameliorated anemia and splenomegaly in / mice, and suppression and PRC2 mutations correlated with the elevated mRNA levels in patients with MPNs, especially myelofibrosis. These findings suggest the crucial role of HMGA2 in MPN progression.
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http://dx.doi.org/10.1182/bloodadvances.2017004457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728313PMC
June 2017

A New Conditionally Immortalized Human Fetal Brain Pericyte Cell Line: Establishment and Functional Characterization as a Promising Tool for Human Brain Pericyte Studies.

Mol Neurobiol 2018 Jul 11;55(7):5993-6006. Epub 2017 Nov 11.

Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, 260-8675, Japan.

While pericytes wrap around microvascular endothelial cells throughout the human body, their highest coverage rate is found in the brain. Brain pericytes actively contribute to various brain functions, including the development and stabilization of the blood-brain barrier (BBB), tissue regeneration, and brain inflammation. Accordingly, detailed characterization of the functional nature of brain pericytes is important for understanding the mechanistic basis of brain physiology and pathophysiology. Herein, we report on the development of a new human brain pericyte cell line, hereafter referred to as the human brain pericyte/conditionally immortalized clone 37 (HBPC/ci37). Developed via the cell conditionally immortalization method, these cells exhibited excellent proliferative ability at 33 °C. However, when cultured at 37 °C, HBPC/ci37 cells showed a differentiated phenotype that was marked by morphological alterations and increases in several pericyte-enriched marker mRNA levels, such as platelet-derived growth factor receptor β. It was also found that HBPC/ci37 cells possessed the facilitative ability of in vitro BBB formation and differentiation into a neuronal lineage. Furthermore, HBPC/ci37 cells exhibited the typical "reactive" features of brain pericytes in response to pro-inflammatory cytokines. To summarize, our results clearly demonstrate that HBPC/ci37 cells possess the ability to perform several key brain pericyte functions while also showing the capacity for extensive and continuous proliferation. Based on these findings, it can be expected that, as a unique human brain pericyte model, HBPC/ci37 cells have the potential to contribute to significant advances in the understanding of human brain pericyte physiology and pathophysiology.
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http://dx.doi.org/10.1007/s12035-017-0815-9DOI Listing
July 2018