Neutrophil elastase is severely down-regulated in severe congenital neutropenia independent of ELA2 or HAX1 mutations but dependent on LEF-1

Skokowa, Julia; Fobiwe, John Paul; Lan, Dan; Thakur, Basant Kumar; Welte, Karl

doi:10.1182/blood-2008-11-188755

Cited by 33 publications

(34 citation statements)

References 40 publications

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“…ELANE mRNA expression in myeloid progenitors and the protein level of NE in plasma are markedly reduced in SCN patients with mutations in ELANE or HAX1. 20 Consistent with this, the proportion of NE-positive cells was significantly lower in blood cells derived from HAX1-iPS cells than in those derived from normal iPS cells ( Figure 2E). Thus, the level of functionally mature neutrophils decreased during in vitro granulopoietic differentiation of HAX1-iPS cells.…”

Section: Maturation Arrest At the Progenitor Level In Neutrophil Diffsupporting

confidence: 72%

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: Maturation Arrest At the Progenitor Level In Neutrophil Diffsupporting

confidence: 72%

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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“…2E). ELANE expression was significantly lower in nonadherent cells derived from SCN-iPS vs. control iPS cells on days 2 and 4 of culture (P < 0.01), as reported (16,17). However, the former was a little higher than the latter on day 7 (P < 0.01).…”

Section: Resultsmentioning

confidence: 70%

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

Hiramoto

Ebihara

Mizoguchi

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The derivation of induced pluripotent stem (iPS) cells from individuals of genetic disorders offers new opportunities for basic research into these diseases and the development of therapeutic compounds. Severe congenital neutropenia (SCN) is a serious disorder characterized by severe neutropenia at birth. SCN is associated with heterozygous mutations in the neutrophil elastase [elastase, neutrophil-expressed (ELANE)] gene, but the mechanisms that disrupt neutrophil development have not yet been clarified because of the current lack of an appropriate disease model. Here, we generated iPS cells from an individual with SCN (SCN-iPS cells). Granulopoiesis from SCN-iPS cells revealed neutrophil maturation arrest and little sensitivity to granulocyte-colony stimulating factor, reflecting a disease status of SCN. Molecular analysis of the granulopoiesis from the SCN-iPS cells vs. control iPS cells showed reduced expression of genes related to the wingless-type mmtv integration site family, member 3a (Wnt3a)/β-catenin pathway [e.g., lymphoid enhancer-binding factor 1], whereas Wnt3a administration induced elevation lymphoid enhancer-binding factor 1-expression and the maturation of SCN-iPS cell-derived neutrophils. These results indicate that SCN-iPS cells provide a useful disease model for SCN, and the activation of the Wnt3a/β-catenin pathway may offer a novel therapy for SCN with ELANE mutation.

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“…Interestingly, all patients, independent of the genetic subtype, demonstrate common downstream pathomechanisms such as lack of LEF1, C/EBPα and ELANE expression. 23,24 Our results also challenge the proposed algorithms for mutational analyses in congenital neutropenia. With the common procedure of first testing the ELANE gene as the most frequent candidate gene in congenital neutropenia, patients #1 and 2 would have been assigned to the group of patients with ELANE as the disease causing gene.…”

Section: New Mutations In G6pc3 and Hax1 Were Not Present In 400 Allementioning

confidence: 77%

Digenic mutations in severe congenital neutropenia

Germeshausen¹,

Zeidler²,

Stuhrmann³

et al. 2010

Haematologica

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Severe congenital neutropenia a clinically and genetically heterogeneous disorder. Mutations in different genes have been described as causative for severe neutropenia, e.g. ELANE, HAX1 and G6PC3. Although congenital neutropenia is considered to be a group of monogenic disorders, the phenotypic heterogeneity even within the yet defined genetic subtypes points to additional genetic and/or epigenetic influences on the disease phenotype. We describe congenital neutropenia patients with mutations in two candidate genes each, including 6 novel mutations. Two of them had a heterozygous ELANE mutation combined with a homozygous mutation in G6PC3 or HAX1, respectively. The other 2 patients combined homozygous or compound heterozygous mutations in G6PC3 or HAX1 with a heterozygous mutation in the respective other gene. Our results suggest that digenicity may underlie this disorder of myelopoiesis at least in some congenital neutropenia patients.

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Neutrophil elastase is severely down-regulated in severe congenital neutropenia independent of ELA2 or HAX1 mutations but dependent on LEF-1

Cited by 33 publications

References 40 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

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

Digenic mutations in severe congenital neutropenia

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