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Gene correction of HAX1 reversed Kostmann disease phenotype in patient-specific induced pluripotent stem cells.

ABSTRACT: Severe congenital neutropenia (SCN, Kostmann disease) is a heritable disorder characterized by a granulocytic maturation arrest. Biallelic mutations in HCLS1 associated protein X-1 (HAX1) are frequently detected in affected individuals, including those of the original pedigree described by Kostmann in 1956. To date, no faithful animal model has been established to study SCN mediated by HAX1 deficiency. Here we demonstrate defective neutrophilic differentiation and compensatory monocyte overproduction from patient-derived induced pluripotent stem cells (iPSCs) carrying the homozygous HAX1W44X nonsense mutation. Targeted correction of the HAX1 mutation using the CRISPR-Cas9 system and homologous recombination rescued neutrophil differentiation and reestablished an HAX1 and HCLS1-centered transcription network in immature myeloid progenitors, which is involved in the regulation of apoptosis, apoptotic mitochondrial changes, and myeloid differentiation. These findings made in isogenic iPSC-derived myeloid cells highlight the complex transcriptional changes underlying Kostmann disease. Thus, we show that patient-derived HAX1W44X -iPSCs recapitulate the Kostmann disease phenotype in vitro and confirm HAX1 mutations as the disease-causing monogenic lesion. Finally, our study paves the way for nonvirus-based gene therapy approaches in SCN.

SUBMITTER: Pittermann E

PROVIDER: S-EPMC5737589 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Gene correction of HAX1 reversed Kostmann disease phenotype in patient-specific induced pluripotent stem cells.

Pittermann Erik E Lachmann Nico N MacLean Glenn G Emmrich Stephan S Ackermann Mania M Göhring Gudrun G Schlegelberger Brigitte B Welte Karl K Schambach Axel A Heckl Dirk D Orkin Stuart H SH Cantz Tobias T Klusmann Jan-Henning JH

Blood advances 20170602 14

Severe congenital neutropenia (SCN, Kostmann disease) is a heritable disorder characterized by a granulocytic maturation arrest. Biallelic mutations in HCLS1 associated protein X-1 (HAX1) are frequently detected in affected individuals, including those of the original pedigree described by Kostmann in 1956. To date, no faithful animal model has been established to study SCN mediated by HAX1 deficiency. Here we demonstrate defective neutrophilic differentiation and compensatory mono ...[more]

PMID: 29296734

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Project description:BackgroundNiemann-Pick type C1 disease (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. In this lysosomal storage disorder the intracellular transport and sequestration of several lipids like cholesterol is severely impaired, resulting in an accumulation of lipids in late endosomes and lysosomes. The neurological manifestation of the disease is caused by dysfunction and cell death in the central nervous system. Several animal models were used to analyze the impaired pathways. However, the underlying pathogenic mechanisms are still not completely understood and the genetic variability in humans cannot be reflected in these models. Therefore, a human model using patient-specific induced pluripotent stem cells provides a promising approach.MethodsWe reprogrammed human fibroblasts from a NPC1 patient and a healthy control by retroviral transduction with Oct4, Klf4, Sox2 and c-Myc. The obtained human induced pluripotent stem cells (hiPSCs) were characterized by immunocytochemical analyses. Neural progenitor cells were generated and patch clamp recordings were performed for a functional analysis of derived neuronal cells. Filipin stainings and the Amplex Red assay were used to demonstrate and quantify cholesterol accumulation.ResultsThe hiPSCs expressed different stem cell markers, e.g. Nanog, Tra-1-81 and SSEA4. Using the embryoid body assay, the cells were differentiated in cells of all three germ layers and induced teratoma in immunodeficient mice, demonstrating their pluripotency. In addition, neural progenitor cells were derived and differentiated into functional neuronal cells. Patch clamp recordings revealed voltage dependent channels, spontaneous action potentials and postsynaptic currents. The accumulation of cholesterol in different tissues is the main hallmark of NPC1. In this study we found an accumulation of cholesterol in fibroblasts of a NPC1 patient, derived hiPSCs, and neural progenitor cells, but not in cells derived from fibroblasts of a healthy individual. These findings were quantified by the Amplex Red assay, demonstrating a significantly elevated cholesterol level in cells derived from fibroblasts of a NPC1 patient.ConclusionsWe generated a neuronal model based on induced pluripotent stem cells derived from patient fibroblasts, providing a human in vitro model to study the pathogenic mechanisms of NPC1 disease.

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Gene correction of HAX1 reversed Kostmann disease phenotype in patient-specific induced pluripotent stem cells.

Publications

Gene correction of <i>HAX1</i> reversed Kostmann disease phenotype in patient-specific induced pluripotent stem cells.

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