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

ABSTRACT: 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.

SUBMITTER: Hiramoto T

PROVIDER: S-EPMC3581925 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Severe congenital neutropenia: genetics and pathogenesis.

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Project description:Accurate and efficient DNA synthesis is an essential function of replicating cells. Mutations in replisome components lead to a number of syndromic diseases including immunodeficiencies. Dumbbell former 4 (DBF4) is the regulatory subunit of the DBF4-dependent kinase (DDK) which is essential for the activation of replication origins. We here studied a patient with severe congenital neutropenia (SCN) and syndromic features without a genetic diagnosis. We identified a private homozygous mutation in DBF4 (CADD 25.8 with a DBF4-specific MSC of 3.13) in a patient with SCN. The DBF4 mutant is normally expressed in stimulated PBMCs and dermal fibroblasts, but has a decreased CDC7-binding capacity in overexpression assays. DDK-specific phosphorylation of MCM2 was decreased in stimulated PBMCs with accumulation of CDK inhibitor p21, a G0 cell cycle arrest and impaired proliferation. Serum-starved fibroblasts showed a similar cell cycle phenotype but no p21 accumulation and normal MCM2 phosphorylation. In vitro differentiation of primary CD34+ cells recapitulated the SCN phenotype observed in vivo and was associated with a 4-fold increase in p21 gene expression. Single cell RNA sequencing of whole bone marrow revealed upregulation of p53 targets and activation of the PERK pathway of the unfolded protein response. Autosomal recessive functional DBF4 deficiency causes SCN with syndromic features.

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

Project description:Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE, which encodes neutrophil elastase (NE). However, a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end, we generated both normal and SCN patient-derived induced pluripotent stem cells (iPSCs), and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest, and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly, high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient-derived iPSCs through C/EBP?-dependent emergency granulopoiesis. In contrast, sivelestat, an NE-specific small-molecule inhibitor, corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA, but not CEBPB; and promoting promyelocyte survival and differentiation. Together, these data suggest that SCN disease pathogenesis includes NE mislocalization, which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization.

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