Publications by authors named "Hideo Ema"

57 Publications

Gene knockout in highly purified mouse hematopoietic stem cells by CRISPR/Cas9 technology.

J Immunol Methods 2021 May 4;495:113070. Epub 2021 May 4.

State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China. Electronic address:

The CRISPR/Cas9 system has been used for genome editing of human and mouse cells. In this study, we established a protocol for gene knockout (KO) in mouse hematopoietic stem cells (HSCs). HSCs were highly purified from the bone marrow of tamoxifen-treated Cas9-EGFP/Cre-ER transgenic mice, maintained in serum-free polyvinyl alcohol culture with cytokines, lentivirally transduced with sgRNA-Crimson, and transplanted into lethally irradiated mice with competitor cells. Previous studies of Pax5 KO mice have shown B cell differentiation block. To verify our KO HSC strategy, we deleted Pax5 gene in 600 CD201CD150CD48c-KitSca-1Lin cells (HSC1 cells), highly enriched in myeloid-biased HSCs, and CD201CD150CD48 c-KitSca-1Lin cells (HSC2 cells), highly enriched in lymphoid-biased HSCs. As predicted, both Pax5 KO HSC1 and HSC2 cells showed few B cells in the peripheral blood and the accumulation of pro-B cells in the bone marrow of recipient mice. Our data suggesetd that myeloid-biased and lymphoid-biased HSCs share a common B cell differentiation pathway. This population-specific KO strategy will find its applications for gene editing in a varity of somatic cells, particuarly rare stem and progenitor cells from different tissues.
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http://dx.doi.org/10.1016/j.jim.2021.113070DOI Listing
May 2021

Analysis and Isolation of Mouse Leukemic Stem Cells.

Methods Mol Biol 2021 ;2185:51-63

Department of Regenerative Medicine, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.

Flow cytometry has been widely used in basic and clinical research for analysis of a variety of normal and malignant cells. Hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) can be highly purified by flow cytometry. Isolated HSCs and LSCs can be functionally identified by transplantation assays and can also be studied at the molecular level. Here we describe the flow cytometry methods for analysis and isolation of mouse HSCs and LSCs.
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http://dx.doi.org/10.1007/978-1-0716-0810-4_4DOI Listing
March 2021

Probing the fate of transplanted hematopoietic stem cells: is the combinational approach "FIT" for purpose?

Sci China Life Sci 2020 Nov 25;63(11):1755-1758. Epub 2020 Aug 25.

State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China.

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http://dx.doi.org/10.1007/s11427-020-1786-4DOI Listing
November 2020

[Hematopoietic Stem Cells Differentiate into the Megakaryocyte Lineage--Review].

Zhongguo Shi Yan Xue Ye Xue Za Zhi 2020 Jun;28(3):1044-1048

State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Disease Hospital, Chinese Academy of Medical, Sciences & Peking Union Medical College, Tianjin 300020, China,E-mail:

Abstract  Hematopoietic stem cells are able to self-renewal and differentiate to all blood lineages. With the development of new technologies, recent studies have proposed the revised versions of hematopoiesis. In the classical model of hematopoietic differentiation, HSCs were located at the apex of hematopoietic hierarchy. During differentiation process, HSCs progressively lose self-renewal potential to be commited to progenitors with restricted differentiation potential. For instance, HSCs first give rise to multipotent progenitor cells, then produce bipotent and unipotent progenitors, and finally differentiate to mature blood cells. For the differentiation of megakaryocytes, common myeloid progenitors derived from HSCs give rise to megakaryocyte-erythrocyte progenitors and then develop to megakaryocytes. However, recent results show that megakaryocytes can be directly generated from HSCs without multipotent or bipotent phases. Alternatively, platelet-biased HSCs produce megakaryocyte progenitors. In this article, recent advances in the hematopoiesis and megakaryocyte differentiation pathway are reviewed.
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http://dx.doi.org/10.19746/j.cnki.issn.1009-2137.2020.03.054DOI Listing
June 2020

Differentiation of transplanted haematopoietic stem cells tracked by single-cell transcriptomic analysis.

Nat Cell Biol 2020 06 4;22(6):630-639. Epub 2020 May 4.

State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.

How transplanted haematopoietic stem cells (HSCs) behave soon after they reside in a preconditioned host has not been studied due to technical limitations. Here, using single-cell RNA sequencing, we first obtained the transcriptome-based classifications of 28 haematopoietic cell types. We then applied them in conjunction with functional assays to track the dynamic changes of immunophenotypically purified HSCs in irradiated recipients within the first week after transplantation. Based on our transcriptional classifications, most homed HSCs in bone marrow and spleen became multipotent progenitors and, occasionally, some HSCs gave rise to megakaryocytic-erythroid or myeloid precursors. Parallel in vitro and in vivo functional experiments supported the paradigm of robust differentiation without substantial HSC expansion during the first week. Therefore, this study uncovers the previously inaccessible kinetics and fate choices of transplanted HSCs in myeloablated recipients at early stage, with implications for clinical applications of HSCs and other stem cells.
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http://dx.doi.org/10.1038/s41556-020-0512-1DOI Listing
June 2020

Granulocyte colony-stimulating factor directly acts on mouse lymphoid-biased but not myeloid-biased hematopoietic stem cells.

Haematologica 2021 Jun 1;106(6):1647-1658. Epub 2021 Jun 1.

Institute of Hematology and Blood Diseases Hospital.

Granulocyte colony-stimulating factor (G-CSF) is widely used in clinical settings to mobilize hematopoietic stem cells (HSCs) into the circulation for HSC harvesting and transplantation. However, whether G-CSF directly stimulates HSCs to change their cell cycle state and fate is controversial. HSCs are a heterogeneous population consisting of different types of HSCs, such as myeloid-biased HSCs and lymphoid-biased HSCs. We hypothesized that G-CSF has different effects on different types of HSCs. To verify this, we performed serum-free single-cell culture and competitive repopulation with cultured cells. Single highly purified HSCs and hematopoietic progenitor cells (HPCs) were cultured with stem cell factor (SCF), SCF + G-CSF, SCF + granulocyte/macrophage (GM)-CSF, or SCF + thrombopoietin (TPO) for 7 days. Compared with SCF alone, SCF + G-CSF increased the number of divisions of cells from the lymphoid-biased HSC-enriched population but not that of cells from the My-bi HSC-enriched population. SCF + G-CSF enhanced the level of reconstitution of lymphoid-biased HSCs but not that of myeloid-biased HSCs. Clonal transplantation assay also showed that SCF + G-CSF did not increase the frequency of myeloid-biased HSCs. These data showed that G-CSF directly acted on lymphoid-biased HSCs but not myeloid-biased HSCs. Our study also revised the cytokine network at early stages of hematopoiesis: SCF directly acted on myeloid-biased HSCs; TPO directly acted on myeloid-biased HSCs and lymphoid-biased HSCs; and GM-CSF acted only on HPCs. Early hematopoiesis is controlled differentially and sequentially by a number of cytokines.
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http://dx.doi.org/10.3324/haematol.2019.239251DOI Listing
June 2021

[Research Advance on In Vitro Generation of Human Hematopoietic Stem Cells for Transplantation--Review].

Zhongguo Shi Yan Xue Ye Xue Za Zhi 2020 Feb;28(1):320-324

State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Disease, Institute of Hematology & Blood Disease Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China,Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, Tianjin 300020, China,E-mail:

Abstract  Currently, hematopoietic stem cell (HSC) transplantation is widely used in the therapy of hematological malignancies, non-malignant refractory anemia, genetic diseases and certain tumors with satisfactory therapeutic efficacy. HSC sources used for transplantation include bone marrow, mobilized peripheral blood and neonate umbilical cord blood. However, for many patients, sufficient number of human leukocyte antigen (HLA) -matched HSC cannot be found for transplantation, because the number of HSC in these tissues is small and HLA-identical donors are rare. Thus, in vitro generation of HSC has recently been focused. At present, the origin of HSC is hPSC, including hESC and hiPSC, which is worth to be the new origin of HSC transplantation. However, to generate functional hematopoietic stem cells which have efficient multi-lineage differentiation and in vivo engraftment potentials still is a big challenge to be confronted. In this review, the recent technical progress in HSC generation is summarizd, and the problems to be solved and new challenges to be confronted were discussed.
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http://dx.doi.org/10.19746/j.cnki.issn.1009-2137.2020.01.053DOI Listing
February 2020

Lineage marker expression on mouse hematopoietic stem cells.

Exp Hematol 2019 08 9;76:13-23.e2. Epub 2019 Jul 9.

State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China; National Clinical Research Center for Hematological disorders, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China; Department of Regenerative Medicine, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China. Electronic address:

Whether hematopoietic stem cells (HSCs) express lineage markers is controversial. In this study, we highly purified HSCs from the adult bone marrow of C57BL/6 mice and examined their gene expression and reconstitution potential. We first focused on the integrin family. Single-cell reverse transcription polymerase chain reaction revealed that the expression of ItgaM/Itgb2 (Mac-1) and Itga2b/Itgb3 (CD41/CD61) gradually increased along HSC differentiation, whereas Itga4, Itga5, Itga6, and ItgaV (CD51) together with Itgb1 were highly expressed in both HSCs and hematopoietic progenitor cells (HPCs). We next fractionated HSCs based on their expression of Mac-1, CD41, and CD51 by flow cytometry. We detected Mac-negative and Mac-low, but not Mac-high cells, in the HSC population. We also detected CD41-negative, -low, and -high cells in the HSC population. Competitive repopulation revealed that Mac-1-negative and -low HSCs were functionally similar, and CD41-negative and -low HSCs were functionally similar, at the single-cell level, but CD41-high HSCs were not detectable. We then found that the selection of Mac-1-negative HSCs or CD41-negative HSCs had no advantage in HSC purification. We moreover found that HSCs expressed more CD51 than CD41, and HPCs expressed more CD41 than CD51, suggesting that CD51 expression was gradually replaced by CD41 expression during megakaryocyte differentiation. We concluded that low levels of Mac-1 and CD41 expression are irrelevant to the self-renewal and differentiation potentials in HSCs.
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http://dx.doi.org/10.1016/j.exphem.2019.07.001DOI Listing
August 2019

[Emerging role of Mitochondria in Maintenance of Hematopoietic Stem Cells--Review].

Zhongguo Shi Yan Xue Ye Xue Za Zhi 2019 Feb;27(1):277-282

State Key Laboratory of Experimental Hematology,Institute of Hematology & Blood Diseases Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College,Tianjin 300020,China.E-mail:

Mitochondria are double-membrane organelles existing only in eukaryotic cells. Mitochondria perform various important functions,such as producing energy,regulating signal transduction,and contributing to stress response. Recent studies have highlighted an important role of mitochondria in the determination of hematopoietic stem cells (HSC) fate. Limited biogenesis or timely clearance of mitochondria is an important way against oxidative stress,which favors the quiescence of HSC. Accumulation of mitochondria may lead to proliferation of HSC,even the aging of HSC. Mitochondrial signaling regulates Ca concentration,which is essential for HSC differentiation. This review summarizes the current findings of the mitochondrial roles in HSC quiescence,self-renewal,lineage differentiation and aging.
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http://dx.doi.org/10.7534/j.issn.1009-2137.2019.01.045DOI Listing
February 2019

Mouse acute leukemia develops independent of self-renewal and differentiation potentials in hematopoietic stem and progenitor cells.

Blood Adv 2019 02;3(3):419-431

State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.

The cell of origin, defined as the normal cell in which the transformation event first occurs, is poorly identified in leukemia, despite its importance in understanding of leukemogenesis and improving leukemia therapy. Although hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) were used for leukemia models, whether their self-renewal and differentiation potentials influence the initiation and development of leukemia is largely unknown. In this study, the self-renewal and differentiation potentials in 2 distinct types of HSCs (HSC1 [CD150CD41CD34LineageSca-1c-Kit cells] and HSC2 [CD150CD41CD34LineageSca-1c-Kit cells]) and 3 distinct types of HPCs (HPC1 [CD150CD41CD34LineageSca-1c-Kit cells], HPC2 [CD150CD41CD34LineageSca-1c-Kit cells], and HPC3 [CD150CD41CD34LineageSca-1c-Kit cells]) were isolated from adult mouse bone marrow, and examined by competitive repopulation assay. Then, cells from each population were retrovirally transduced to initiate MLL-AF9 acute myelogenous leukemia (AML) and the intracellular domain of NOTCH-1 T-cell acute lymphoblastic leukemia (T-ALL). AML and T-ALL similarly developed from all HSC and HPC populations, suggesting multiple cellular origins of leukemia. New leukemic stem cells (LSCs) were also identified in these AML and T-ALL models. Notably, switching between immunophenotypical immature and mature LSCs was observed, suggesting that heterogeneous LSCs play a role in the expansion and maintenance of leukemia. Based on this mouse model study, we propose that acute leukemia arises from multiple cells of origin independent of the self-renewal and differentiation potentials in hematopoietic stem and progenitor cells and is amplified by LSC switchover.
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http://dx.doi.org/10.1182/bloodadvances.2018022400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373745PMC
February 2019

[Advances of studies on Purification and Tracking of Hematopoietic Stem Cells Using Their Specific Gene Expression--Review].

Zhongguo Shi Yan Xue Ye Xue Za Zhi 2018 Aug;26(4):1215-1219

State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020; Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin 300020; Department of Stem Cell and Regenerative Medicine, Peking Union Medical College, Tianjin 300020, China .E-mail:

Hematopoietic stem cells (HSC) maintain homeostatic hematopoiesis via their multi-lineage differentiation and self-renewal potentials. HSC can be enriched and purified by flow cytometry relying on their cell surface markers and functional characteristics, however, these methods can not meet the need for deep analysis of HSC biological property and function because of the poor purity. Recent studies have successfully purified and tracked HSC using specifically expressed genes, which can enhance the purification efficiency of HSC, thus provide a better tool for the in-vivo study of HSC. This review summarizes the new techniques and discusses their advantages and disadvantages.
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http://dx.doi.org/10.7534/j.issn.1009-2137.2018.04.045DOI Listing
August 2018

[Relationship between Early Peak Temperature and Neutropenia Duration in Acute Leukemia Patients after Chemotherapy and Its Mechanism].

Zhongguo Shi Yan Xue Ye Xue Za Zhi 2018 Jun;26(3):665-670

Institute of Hematology and Blood Diseases Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College,Tianjin 300020, China. E-mail:

Objective: To investigate the relationship between early peak body temperature and neutropenia duration and its potential mechanism.

Methods: A total of 111 patients with CR1 phase acute leukemia (AL) with neutropenia infection were enrolled in this study. The relationship between early peak body temperature and neutropenia duration was analyzed retrospectively, and the IL-6 serum level in patients with different peak of body temperature was detected, and the single cell culture system in vitro was established, the incorparation rate of EdU in vivo was detected, and the effect of IL-6 on mouse hematopoietic stem cells /progenitor cells was analyzed.

Results: Out of 111 patients with nentropenia, the body temperature <38 °C and the neutropenia duration 9.5±3.69 d were observed in 44 patients, while the body temperature >38 °C and neutropenia duration 7.33±4.20 d were observed in 69 patients, the differences between 2 groups was statistically signficant (P<0.05). The EdU test showed that the number of EdU hematopoietic stem cells and progenitor cells increased. The IL-6 level was different in patients with different peaks of initial bady temperature (P<0.05). The results of amimal experiment showed that the IL-6 could promote the proliferation of hematopoietic stem cells/ progenitor cells in vitro and in vivo.

Conclusion: For patients with neutropenic infection when initial body temperature peak is <38 °C, the probability of neutropenia duration prolonging after chamotherapy increases, which may relate with promotive effect of pro-inflammatory cytokins on prliferation of hematopoietic stem cells/progenitor cells.
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http://dx.doi.org/10.7534/j.issn.1009-2137.2018.03.006DOI Listing
June 2018

[Hematopoietic Stem Cells or Hematopoietic Progenitor Cells Maintain the Steady-State Hematopoiesis? -Editorial].

Zhongguo Shi Yan Xue Ye Xue Za Zhi 2018 Jun;26(3):637-641

State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Disease Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China; Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin 300020, China; Department of Stem Cells and Regenerative Medicine, Peking Union Medical College, Tianjin 300020, China. E-mail:

Current dogma suggests that hematopoietic stem cells (HSC) reside in the top of the hematopoietic hierarchy, which can provide all kinds of mature blood cells constantly through self-renewal and multilineage differentiation potential. HSC has been regarded as the main cell population that maintains the stable hematopoiesis and several differentiation and development patterns of HSC have been summarized based on transplantation results. However, in deed the transplantation experiment is based on an extremely situation of stress which could not really reflect the function of HSC in normal homeostatic condition. Recent studies show that hematopoietic progenitor cells (HPC) play the most important role in hematopoiesis based on different experimental strategies. This article focuses on the controversial subject of the function of HSC and HPC under homostasis.
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http://dx.doi.org/10.7534/j.issn.1009-2137.2018.03.001DOI Listing
June 2018

TGF-β1 Negatively Regulates the Number and Function of Hematopoietic Stem Cells.

Stem Cell Reports 2018 07 21;11(1):274-287. Epub 2018 Jun 21.

State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing Road 288, Tianjin 300020, China. Electronic address:

Transforming growth factor β1 (TGF-β1) plays a role in the maintenance of quiescent hematopoietic stem cells (HSCs) in vivo. We asked whether TGF-β1 controls the cell cycle status of HSCs in vitro to enhance the reconstitution activity. To examine the effect of TGF-β1 on the HSC function, we used an in vitro culture system in which single HSCs divide with the retention of their short- and long-term reconstitution ability. Extensive single-cell analyses showed that, regardless of its concentration, TGF-β1 slowed down the cell cycle progression of HSCs but consequently suppressed their self-renewal potential. Cycling HSCs were not able to go back to quiescence with TGF-β1. This study revealed a negative role of TGF-β1 in the regulation of the HSC number and reconstitution activity.
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http://dx.doi.org/10.1016/j.stemcr.2018.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067088PMC
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

PDK1 plays a vital role on hematopoietic stem cell function.

Sci Rep 2017 07 10;7(1):4943. Epub 2017 Jul 10.

State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, and Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a pivotal regulator in the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway that have been shown to play key roles in the functional development of B and T cells via activation of AGC protein kinases during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. Here we specifically deleted the PDK1 gene in the hematopoietic system and found that PDK1-deficient HSCs exhibited impaired function and defective lineage commitment abilities. Lack of PDK1 caused HSCs to be less quiescent and to produce a higher number of phenotypic HSCs and fewer progenitors. PDK1-deficient HSCs were also unable to reconstitute the hematopoietic system. Notably, HSC function was more dependent on PDK1 than on mTORC2, which indicates that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. PDK1-deficient HSCs also exhibited reduced ROS levels, and treatment of PDK1-deficient HSCs with L-butathioninesulfoximine in vitro elevated the low ROS level and promoted colony formation. Therefore, PDK1 appears to contribute to HSC function partially via regulating ROS levels.
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http://dx.doi.org/10.1038/s41598-017-05213-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5504031PMC
July 2017

An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors.

Stem Cell Reports 2017 03 23;8(3):500-508. Epub 2017 Feb 23.

Laboratory of Stem Cell Therapy, Center for Experimental Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan; Project Division of Advanced Regenerative Medicine, The Institute of Medical Science, the University of Tokyo, Tokyo 108-8639, Japan. Electronic address:

Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.
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http://dx.doi.org/10.1016/j.stemcr.2017.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355634PMC
March 2017

Chasing the precursor of functional hematopoietic stem cells at the single cell levels in mouse embryos.

J Hematol Oncol 2016 07 22;9(1):58. Epub 2016 Jul 22.

State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, 300020, China.

Background: Adult hematopoietic stem cells (HSCs), the ideal system for regenerative research, were isolated at single cell levels decades ago, whereas studies on embryonic HSCs are much more difficult.

Methods: Zhou et al identified a new pre-HSC cell surface marker, CD201, by which they isolated pre-HSCs at single cell levels for further analyses.

Results: The novel expression pattern of HSC development is revealed, including the fundamental role of mammalian targets of rapamycin (mTOR) signaling pathway in HSCs emergence, and the repopulation potential of S/G2/M phase pre-HSCs.

Conclusions: Deeper understandings of the cellular origin and developmental regulatory network of HSCs are essential to develop new strategies of generating HSCs in vitro for clinical application.
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http://dx.doi.org/10.1186/s13045-016-0289-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4957898PMC
July 2016

A novel lymphoid progenitor cell population (LSK(low)) is restricted by p18(INK4c).

Exp Hematol 2016 09 8;44(9):874-885.e5. Epub 2016 Jun 8.

State Key Laboratory of Experimental Hematology, Tianjin, China; Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China; Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin, China; Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, Tianjin, China; Collaborative Innovation Center for Cancer Medicine, Tianjin, China. Electronic address:

The cyclin-dependent kinase inhibitor CDKN2C (p18(INK4c)) restrains self-renewal in hematopoietic stem cells (HSCs) and participates in the development and maturation of lymphoid cells. Deficiency in p18 predisposes mice and humans to hematopoietic lymphoid malignancies such as T-cell leukemia and multiple myeloma. However, the mechanism by which p18 regulates differentiation from HSCs to lymphoid cells is poorly understood. In this study, we found that a progenitor population characterized by its expression of surface markers, Lin(-) Sca-1(+) c-Kit(low) (LSK(low)), was markedly expanded in the bone marrow of p18 knock-out (p18(-/-)) mice. This novel population possessed lymphoid differentiation potential, but not myeloid differentiation potential, both in vitro and in vivo. Whereas LSK(low) cells and common lymphoid progenitors (CLPs) overlapped functionally in generating lymphoid cells, they were distinct cell populations, because they had different gene expression profiles. Unlike CLPs, LSK(low) cells did not express the interleukin-7 receptor. LSK(low) cells were derived from HSCs and were independent of the p18-deleted microenvironment. This cell population may represent a previously unappreciated transitional stage from HSCs to lymphoid progenitors that is strictly restricted by p18 under physiological conditions. Likewise, LSK(low) might serve as a new cellular target of lymphoid malignances in the absence of p18.
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http://dx.doi.org/10.1016/j.exphem.2016.05.015DOI Listing
September 2016

Promotion of Expansion and Differentiation of Hematopoietic Stem Cells by Interleukin-27 into Myeloid Progenitors to Control Infection in Emergency Myelopoiesis.

PLoS Pathog 2016 Mar 18;12(3):e1005507. Epub 2016 Mar 18.

Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan.

Emergency myelopoiesis is inflammation-induced hematopoiesis to replenish myeloid cells in the periphery, which is critical to control the infection with pathogens. Previously, pro-inflammatory cytokines such as interferon (IFN)-α and IFN-γ were demonstrated to play a critical role in the expansion of hematopoietic stem cells (HSCs) and myeloid progenitors, leading to production of mature myeloid cells, although their inhibitory effects on hematopoiesis were also reported. Therefore, the molecular mechanism of emergency myelopoiesis during infection remains incompletely understood. Here, we clarify that one of the interleukin (IL)-6/IL-12 family cytokines, IL-27, plays an important role in the emergency myelopoiesis. Among various types of hematopoietic cells in bone marrow, IL-27 predominantly and continuously promoted the expansion of only Lineage-Sca-1+c-Kit+ (LSK) cells, especially long-term repopulating HSCs and myeloid-restricted progenitor cells with long-term repopulating activity, and the differentiation into myeloid progenitors in synergy with stem cell factor. These progenitors expressed myeloid transcription factors such as Spi1, Gfi1, and Cebpa/b through activation of signal transducer and activator of transcription 1 and 3, and had enhanced potential to differentiate into migratory dendritic cells (DCs), neutrophils, and mast cells, and less so into macrophages, and basophils, but not into plasmacytoid DCs, conventional DCs, T cells, and B cells. Among various cytokines, IL-27 in synergy with the stem cell factor had the strongest ability to augment the expansion of LSK cells and their differentiation into myeloid progenitors retaining the LSK phenotype over a long period of time. The experiments using mice deficient for one of IL-27 receptor subunits, WSX-1, and IFN-γ revealed that the blood stage of malaria infection enhanced IL-27 expression through IFN-γ production, and the IL-27 then promoted the expansion of LSK cells, differentiating and mobilizing them into spleen, resulting in enhanced production of neutrophils to control the infection. Thus, IL-27 is one of the limited unique cytokines directly acting on HSCs to promote differentiation into myeloid progenitors during emergency myelopoiesis.
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http://dx.doi.org/10.1371/journal.ppat.1005507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4798290PMC
March 2016

Guest editorial: Institute of Hematology and Blood Diseases Hospital in China.

Authors:
Hideo Ema

Int J Hematol 2016 May 10;103(5):486. Epub 2016 Mar 10.

Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 288 Nanjing Rd., Tianjin, 300020, China.

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http://dx.doi.org/10.1007/s12185-016-1967-5DOI Listing
May 2016

Repopulation dynamics of single haematopoietic stem cells in mouse transplantation experiments: Importance of stem cell composition in competitor cells.

J Theor Biol 2016 Apr 20;394:57-67. Epub 2016 Jan 20.

Department of Biology, Faculty of Sciences, Kyushu University, Japan.

The transplantation of blood tissues from bone marrow into a lethally irradiated animal is an experimental procedure that is used to study how the blood system is reconstituted by haematopoietic stem cells (HSC). In a competitive repopulation experiment, a lethally irradiated mouse was transplanted with a single HSC as a test cell together with a number of bone marrow cells as competitor cells, and the fraction of the test cell progeny (percentage of chimerism) was traced over time. In this paper, we studied the stem cell kinetics in this experimental procedure. The balance between symmetric self-renewal and differentiation divisions in HSC determined the number of cells which HSC produce and the length of time for which HSC live after transplantation. The percentage of chimerism depended on the type of test cell (long-, intermediate-, or short-term HSC), as well as the type and number of HSC included in competitor cells. We next examined two alternative HSC differentiation models, one-step and multi-step differentiation models. Although these models differed in blood cell production, the percentage of chimerism appeared very similar. We also estimated the numbers of different types of HSC in competitor cells. Based on these results, we concluded that the experimental results inevitably include stochasticity with regard to the number and the type of HSC in competitor cells, and that, in order to detect different types of HSC, an appropriate number of competitor cells needs to be used in transplantation experiments.
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http://dx.doi.org/10.1016/j.jtbi.2016.01.010DOI Listing
April 2016

Mechanisms of self-renewal in hematopoietic stem cells.

Authors:
Zhao Wang Hideo Ema

Int J Hematol 2016 May 12;103(5):498-509. Epub 2015 Dec 12.

Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 288 Nanjing Rd, Tianjin, 300020, China.

A large number of studies have shown that quiescence is essential for hematopoietic stem cells (HSCs) to maintain their number and function. Otherwise, HSCs are exhausted or damaged by various substances. We need to understand how the quiescent state is maintained in HSCs, how HSCs are driven into the cell cycle, and how HSCs return to the quiescent state. We also need to understand how cycling HSCs make the decision whether to self-renew. A number of molecules have been reported as candidate regulators of these events in HSCs. In this review, we focus on the HSC niche, the cytokine network, and associated transcription factors; and then discuss to what extent we can currently understand these critical issues in stem cell biology.
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http://dx.doi.org/10.1007/s12185-015-1919-5DOI Listing
May 2016

Heterogeneity and hierarchy of hematopoietic stem cells.

Exp Hematol 2014 Feb 20;42(2):74-82.e2. Epub 2013 Nov 20.

Department of Cell Differentiation, Sakaguchi Laboratories of Developmental Biology, Keio University School of Medicine, Tokyo, Japan.

Hematopoietic stem cells (HSCs) are a more heterogeneous population than previously thought. Extensive analysis of reconstitution kinetics after transplantation allows a new classifications of HSCs based on lineage balance. Previously unrecognized classes of HSCs, such as myeloid- and lymphoid-biased HSCs, have emerged. However, varying nomenclature has been used to describe these cells, promoting confusion in the field. To establish a common nomenclature, we propose a reclassification of short-, intermediate-, and long-term (ST, IT, and LT) HSCs defined as: ST < 6 months, IT > 6 months, and LT > 12. We observe that myeloid-biased HSCs or α cells overlap with LT-HSCs, whereas lymphoid-biased HSCs or γ/δ cells overlap with ST-HSCs, suggesting that HSC lifespan is linked to cell differentiation. We also suggest that HSC heterogeneity prompts reconsideration of long-term (>4 months) multilineage reconstitution as the gold standard for HSC detection. In this review, we discuss relationships among ST-, IT-, and LT-HSCs relevant to stem cell heterogeneity, hierarchical organization, and differentiation pathways.
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http://dx.doi.org/10.1016/j.exphem.2013.11.004DOI Listing
February 2014

Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.

Cell 2013 Aug;154(5):1112-1126

Division of Stem Cell Therapy, Center for Stem Cell Biology and Regeneration Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; Japan Science Technology Agency, ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Electronic address:

Consensus holds that hematopoietic stem cells (HSCs) give rise to multipotent progenitors (MPPs) of reduced self-renewal potential and that MPPs eventually produce lineage-committed progenitor cells in a stepwise manner. Using a single-cell transplantation system and marker mice, we unexpectedly found myeloid-restricted progenitors with long-term repopulating activity (MyRPs), which are lineage-committed to megakaryocytes, megakaryocyte-erythroid cells, or common myeloid cells (MkRPs, MERPs, or CMRPs, respectively) in the phenotypically defined HSC compartment together with HSCs. Paired daughter cell assays combined with transplantation revealed that HSCs can give rise to HSCs via symmetric division or directly differentiate into MyRPs via asymmetric division (yielding HSC-MkRP or HSC-CMRP pairs). These myeloid bypass pathways could be essential for fast responses to ablation stress. Our results show that loss of self-renewal and stepwise progression through specific differentiation stages are not essential for lineage commitment of HSCs and suggest a revised model of hematopoietic differentiation.
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http://dx.doi.org/10.1016/j.cell.2013.08.007DOI Listing
August 2013

Generation of transgenic mouse line expressing Kusabira Orange throughout body, including erythrocytes, by random segregation of provirus method.

Biochem Biophys Res Commun 2013 Jun 17;435(4):586-91. Epub 2013 May 17.

Division of Stem Cell Therapy, 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.

Fluorescent-protein transgenic mice are useful for obtaining marked somatic cells to study kinetics of development or differentiation. Fluorescence-marked hematopoietic stem cells in particular are commonly used for studying hematopoiesis. However, as far as we know, no transgenic mouse line is described in which a fluorescent protein is stably and constitutively expressed in all hematopoietic cells, including erythrocytes and platelets. Using the random segregation of provirus (RSP) method, we generated from retrovirally transduced mouse embryonic stem cells a transgenic mouse line expressing a red/orange fluorescent protein, Kusabira Orange (KuO). KuO transgenic mouse line cells carry only one proviral integration site and stably express KuO in all hematopoietic-lineage elements, including erythrocytes and platelets. Moreover, bone-marrow transplantation in KuO transgenic mice demonstrated normal hematopoieisis. KuO transgenic mice likely will prove useful for study of hematopoiesis that includes erythropoiesis and megakaryopoiesis.
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http://dx.doi.org/10.1016/j.bbrc.2013.05.017DOI Listing
June 2013

Two anatomically distinct niches regulate stem cell activity.

Blood 2012 Sep 11;120(11):2174-81. Epub 2012 Jul 11.

Department of Cell Differentiation, Sakaguchi Laboratory of Developmental Biology, Keio University School of Medicine, Tokyo, Japan.

The niche microenvironment controls stem cell number, fate, and behavior. The bone marrow, intestine, and skin are organs with highly regenerative potential, and all produce a large number of mature cells daily. Here, focusing on adult stem cells in these organs, we compare the structures and cellular components of their niches and the factors they produce. We then define the niche as a functional unit for stem cell regulation. For example, the niche possibly maintains quiescence and regulates fate in stem cells. Moreover, we discuss our hypothesis that many stem cell types are regulated by both specialized and nonspecialized niches, although hematopoietic stem cells, as an exception, are regulated by a nonspecialized niche only. The specialized niche is composed of 1 or a few types of cells lying on the basement membrane in the epithelium. The nonspecialized niche is composed of various types of cells widely distributed in mesenchymal tissues. We propose that the specialized niche plays a role in local regulation of stem cells, whereas the nonspecialized niche plays a role in relatively broad regional or systemic regulation. Further work will verify this dual-niche model to understand mechanisms underlying stem cell regulation.
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http://dx.doi.org/10.1182/blood-2012-04-424507DOI Listing
September 2012

Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche.

Cell 2011 Nov;147(5):1146-58

Japan Science and Technology Agency, ERATO, Chiyoda-ku, Tokyo 102-0075, Japan.

Hematopoietic stem cells (HSCs) reside and self-renew in the bone marrow (BM) niche. Overall, the signaling that regulates stem cell dormancy in the HSC niche remains controversial. Here, we demonstrate that TGF-β type II receptor-deficient HSCs show low-level Smad activation and impaired long-term repopulating activity, underlining the critical role of TGF-β/Smad signaling in HSC maintenance. TGF-β is produced as a latent form by a variety of cells, so we searched for those that express activator molecules for latent TGF-β. Nonmyelinating Schwann cells in BM proved responsible for activation. These glial cells ensheathed autonomic nerves, expressed HSC niche factor genes, and were in contact with a substantial proportion of HSCs. Autonomic nerve denervation reduced the number of these active TGF-β-producing cells and led to rapid loss of HSCs from BM. We propose that glial cells are components of a BM niche and maintain HSC hibernation by regulating activation of latent TGF-β.
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http://dx.doi.org/10.1016/j.cell.2011.09.053DOI Listing
November 2011

Homeostasis of hematopoietic stem cells regulated by the myeloproliferative disease associated-gene product Lnk/Sh2b3 via Bcl-xL.

Exp Hematol 2012 Feb 18;40(2):166-74.e3. Epub 2011 Nov 18.

Laboratory of Stem Cell Therapy, Center for Experimental Medicine, The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan.

Hematopoietic stem cells (HSCs) are maintained at a very low frequency in adult bone marrow under steady-state conditions. However, it is not fully understood how homeostasis of bone marrow HSCs is maintained. We attempted to identify a key molecule involved in the regulation of HSC numbers, a factor that, in the absence of Lnk, leads to HSC expansion. Here, we demonstrate that upon stimulation with thrombopoietin, expression of Bcl-xL, an antiapoptotic protein, was highly enhanced in Lnk-deficient HSCs compared to normal HSCs. As a result, Lnk-deficient HSCs underwent reduced apoptosis following exposure to lethal radiation. Downregulation of Bcl-xL expression in Lnk-deficient HSCs by short-hairpin RNA resulted in a great reduction of their capacity for reconstitution. These findings suggest that Lnk/Sh2b3 constrains the expression of Bcl-xL and that the loss of Lnk/Sh2b3 function enhances survival of HSCs by inhibiting apoptosis. Furthermore, our observations indicate that HSCs in patients with an Lnk/Sh2b3 mutation might become resistant to apoptosis due to thrombopoietin-mediated enhanced expression of Bcl-xL. Consequently, reduced apoptosis could facilitate accumulation of HSCs with oncogenic mutations leading to development of myeloproliferative disorders.
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http://dx.doi.org/10.1016/j.exphem.2011.11.003DOI Listing
February 2012

Functional characterization of hematopoietic stem cells in the spleen.

Exp Hematol 2011 Mar 24;39(3):351-359.e3. Epub 2010 Dec 24.

Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Tokyo, Japan.

Objective: Hematopoietic stem cells (HSCs) reside in both bone marrow (BM) and spleen in adult mice. However, whether BM and spleen HSCs are functionally similar is not known. Spleen HSCs were compared with BM HSCs by various assays.

Materials And Methods: Whole BM and spleen cells were quantitatively analyzed by competitive repopulation. Single-cell transplantation was performed with HSCs purified from BM and spleen. A parabiosis model was used to distinguish organ-specific HSCs from circulating HSCs. The cell cycle was analyzed with pyronin Y staining and bromodeoxyuridine uptake.

Results: Repopulating and self-renewal potentials were similar on a clonal basis between BM and spleen HSCs, whereas the HSC frequency in the spleen was significantly lower than that in the BM. Analysis of parabiotic mice suggested that most HSCs are long-term residents in each organ. Cell-cycle analysis revealed that spleen HSCs cycle twice as frequently as do BM HSCs, suggesting that G(0) phase length is longer in BM HSCs than in spleen HSCs. The cycling difference between BM and spleen HSCs was also observed in mice that had been reconstituted with BM or spleen cells, suggesting that HSC quiescence is regulated in an organ-specific manner.

Conclusions: Spleen HSCs and BM HSCs are functionally similar, but their cycling behaviors differ.
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http://dx.doi.org/10.1016/j.exphem.2010.12.008DOI Listing
March 2011