Wnt3a stimulates maturation of impaired neutrophils developed from severe congenital neutropenia patient-derived pluripotent stem cells

Hiramoto, Takafumi; Ebihara, Yasuhiro; Mizoguchi, Yoko; Nakamura, Kazuhiro; Yamaguchi, Kiyoshi; Ueno, Kazuko; Nariai, Naoki; Mochizuki, Seibu; Yamamoto, Shohei; Nagasaki, Masao; Furukawa, Yoichi; Tani, Kenzaburo; Nakauchi, Hiromitsu; Kobayashi, Masao; Tsuji, Kohichiro

doi:10.1073/pnas.1217039110

Cited by 38 publications

(28 citation statements)

References 29 publications

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“…Defective granulopoiesis was recently reported in SCN-iPS cells with a mutation in ELANE. 24 Our data showing defective granulopoiesis and reduced response to G-CSF administration are generally consistent with this report. The slight differences in CFU-G/GM colony-forming potential between this previous study and the current study might be due to differences in the causative gene (HAX1 or ELANE) or the culture system used for neutrophil differentiation, and/or to variation in the differentiation capabilities of the clones.…”

Section: Discussionsupporting

confidence: 92%

Genetic correction of HAX1 in induced pluripotent stem cells from a patient with severe congenital neutropenia improves defective granulopoiesis

et al. 2013

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T. Morishima et al. 20haematologica | 2014; 99(1) tion system from human iPS cells 17 as well as a serum-and feeder-free monolayer hematopoietic culture system from human ES and iPS cells. 18 In this study, we generate iPS cell lines from an SCN patient with HAX1 gene deficiency and differentiate them into neutrophils in vitro. Furthermore, we corrected for the HAX1 gene deficiency in HAX1-iPS cells by lentiviral transduction with HAX1 cDNA and analyzed the neutrophil differentiation potential of these cells. Thus, this in vitro neutrophil differentiation system from patient-derived iPS cells may be a useful model for future studies in SCN patients with HAX1 gene deficiency. Methods Human iPS cell generationSkin biopsy specimens were obtained from an 11-year old male SCN patient with HAX1 gene deficiency. 19 This study was approved by the Ethics Committee of Kyoto University, and informed consent was obtained from the patient's guardians in accordance with the Declaration of Helsinki. Fibroblasts were expanded in DMEM (Nacalai Tesque, Inc., Kyoto, Japan) containing 10% FBS (vol/vol, Invitrogen, Carlsbad, CA, USA) and 0.5% penicillin and streptomycin (wt/vol, Invitrogen). Generation of iPS cells was performed as described previously. 12 In brief, we introduced OCT3/4, SOX2, KLF4, and cMYC using ecotropic retroviral transduction into patient's fibroblasts expressing mouse Slc7a1. Six days after transduction, cells were harvested and re-plated onto mitotically inactive SNL feeder cells. On the following day, DMEM was replaced with primate ES cell medium (ReproCELL, Kanagawa, Japan) supplemented with basic fibroblast growth factor (5 ng/mL, R&D Systems, Minneapolis, MN, USA). Three weeks later, individual colonies were isolated and expanded. Maintenance of cellsControl ES (KhES-1) and control iPS (253G4 and 201B6) cells were kindly provided by Drs. Norio Nakatsuji and Shinya Yamanaka (Kyoto University, Kyoto, Japan), respectively. These human ES and iPS cell lines were maintained on mitomycin-C (Kyowa Hakko Kirin, Tokyo, Japan)-treated SNL feeder cells as described previously 17 and subcultured onto new SNL feeder cells every seven days. Flow cytometric analysisCells were stained with antibodies as reported previously. 17Samples were analyzed using an LSR flow cytometer and Cell Quest software (Becton-Dickinson). Neutrophil differentiation of iPS cellsIn a previous study, we established a serum and feeder-free monolayer hematopoietic culture system from human ES and iPS cells. 18 In this study, we modified this culture system to direct neutrophil differentiation. iPS cell colonies were cultured on growth factor-reduced Matrigel (Becton-Dickinson)-coated cell culture dishes in Stemline II hematopoietic stem cell expansion medium (Sigma-Aldrich, St. Louis, MO, USA) containing the insulin-transferrin-selenium (ITS) supplement (Invitrogen) and cytokines. iPS cells were treated with cytokines as follows: bone morphogenetic protein (BMP) 4 (20 ng/mL, R&D Systems) was added for four days and then replaced with vas...

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Section: Discussionsupporting

confidence: 92%

Genetic correction of HAX1 in induced pluripotent stem cells from a patient with severe congenital neutropenia improves defective granulopoiesis

et al. 2013

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“…The use of iPSCs to address mechanisms of disease in SCN has been documented by our group and other groups (21,22,43). In this report, we first demonstrate that SCN-associated single point mutations in one allele of ELANE are necessary to induce granulopoietic differentiation arrest and apoptosis of granulocytic progenitors and precursors.…”

Section: Discussionmentioning

confidence: 52%

“…In SCN, the use of iPSCs has provided evidence of canonical Wnt signaling in disease pathogenesis (21). However, a lack of targeted therapies against Wnt signaling has limited the clinical application of this knowledge in the therapy of ELANE-mutant SCN.…”

Section: Cd34mentioning

confidence: 99%

Pathogenesis of ELANE-mutant severe neutropenia revealed by induced pluripotent stem cells

Nayak¹,

Trump²,

Aronow³

et al. 2015

J. Clin. Invest.

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“…Although it is still early days for these models, these lines demonstrate the salient features of SCN, including the promyelocyte-maturation arrest. 52,53 They may thus serve as the preferred models to develop protocols for genetic correction by genome editing and to study the consequences of the combinations of mutations associated with malignant transformation.…”

Section: Discussionmentioning

confidence: 99%

Game of clones: the genomic evolution of severe congenital neutropenia

Touw

2015

Hematology

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Severe congenital neutropenia (SCN) is a genetically heterogeneous condition of bone marrow failure usually diagnosed in early childhood and characterized by a chronic and severe shortage of neutrophils. It is now well-established that mutations in HAX1 and ELANE (and more rarely in other genes) are the genetic cause of SCN. In contrast, it has remained unclear how these mutations affect neutrophil development. Innovative models based on induced pluripotent stem cell technology are being explored to address this issue. These days, most SCN patients receive life-long treatment with granulocyte colony-stimulating factor (G-CSF, CSF3). CSF3 therapy has greatly improved the life expectancy of SCN patients, but also unveiled a high frequency of progression toward myelodysplastic syndrome (MDS) and therapy refractory acute myeloid leukemia (AML). Expansion of hematopoietic clones with acquired mutations in the gene encoding the G-CSF receptor (CSF3R) is regularly seen in SCN patients and AML usually descends from one of these CSF3R mutant clones. These findings raised the questions how CSF3R mutations affect CSF3 responses of myeloid progenitors, how they contribute to the pre-leukemic state of SCN, and which additional events are responsible for progression to leukemia. The vast (sub)clonal heterogeneity of AML and the presence of AML-associated mutations in normally aged hematopoietic clones make it often difficult to determine which mutations are responsible for the leukemic process. Leukemia predisposition syndromes such as SCN are unique disease models to identify the sequential acquisition of these mutations and to interrogate how they contribute to clonal selection and leukemic evolution. Learning Objectives• Knowledge of the possible mechanisms by which mutations in ELANE or HAX1 cause SCN and the controversies that preclude a unifying hypothesis • Understanding of how somatic mutations in CSF3R affect CSF3-induced signal transduction and contribute to the expansion of hematopoietic clones in SCN

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Wnt3a stimulates maturation of impaired neutrophils developed from severe congenital neutropenia patient-derived pluripotent stem cells

Cited by 38 publications

References 29 publications

Genetic correction of HAX1 in induced pluripotent stem cells from a patient with severe congenital neutropenia improves defective granulopoiesis

Genetic correction of HAX1 in induced pluripotent stem cells from a patient with severe congenital neutropenia improves defective granulopoiesis

Pathogenesis of ELANE-mutant severe neutropenia revealed by induced pluripotent stem cells

Game of clones: the genomic evolution of severe congenital neutropenia

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