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

Abstract

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...

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

“…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.…”

Game of clones: the genomic evolution of severe congenital neutropenia

“…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.…”

Severe congenital neutropenias

“…Author Manuscript Author Manuscript iPSCs have been generated from somatic cells of patients with either ELANE or HAX1 mutations [129][130][131] . With the establishment of efficient protocols for in vitro haematopoietic and myeloid differentiation of iPSCs cells 132,133 , these cells could be used as an experimental model to study the pathophysiology of disease.…”