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

Nayak RC, Trump LR, Aronow BJ, Myers K, Mehta P, Kalfa T, Wellendorf AM, Valencia CA, Paddison PJ, Horwitz MS, Grimes HL, Lutzko C, Cancelas JA
Source: J Clin Invest
Publication Date: (2015)
Issue: 125(8): 3103-16
Research Area:
Immunotherapy / Hematology
Stem Cells
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Culture Media:
4D-Nucleofector® X-Unit
For genome editing, iPSCs were cultured on 6-well Matrigel-coated dishes in mTeSR1 until confluence (StemCell Technologies Inc.). Cells were dissociated into single cells using accutase (Sigma-Aldrich) and nucleofected using the Amaxa Nucleofector (Lonza) and kit P3 according to manufacturer’s instructions with 1 µg CRISPR and 1 µg donor construct. After nucleofection, cells were plated on Matrigel-coated dishes in mTeSR1 media supplemented with 10 µM ROCK inhibitor Y-27632 (Millipore). Cells were allowed to expand for 2–3 weeks after cell sorting to recover. Cells were then dissociated into single cells using accutase, and the GFP+ (donor-expressing) cells were sorted by FACS. GFP+ iPSCs were then plated onto Matrigel-coated culture dishes in mTeSR media supplemented with 10 µM ROCK inhibitor Y-27632 for 24 hours. Once iPSC sub-lines were established, iPSCs were transitioned to MEF culture for hematopoietic differentiation.
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/EBPß-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.