Myelopoiesis From Induced Pluripotent Stem Cells Reveals The Role Of Elastase Activity In The Pathogenesis Of Severe Congenital Neutropenia

Severe congenital neutropenia (SCN) is a hereditary neutropenia characterized by granulocytic precursor differentiation arrest in which the pathogenic mechanism is poorly understood. Over half of SCN patients associate with autosomal dominant point mutations in the gene encoding neutrophil elastase (ELANE). High-dose G-CSF therapy succeeds in increasing neutrophil counts in many SCN patients, but the molecular explanation for high dose G-CSF rescue is unknown. Mutations in the G-CSF receptor (CSF3R) associated with a dramatic risk for transformation provides a cautionary note for high dose G-CSF treatment. The pathogenic mechanism of ELANE mutations is incompletely understood due to the lack of disease recapitulation in murine models of ELANE-mutant hematopoiesis and the difficulty to obtain primary, relevant myeloid cell populations from patients. Using induced pluripotent stem cell (iPSC) derived myelopoietic cultures from peripheral blood mononuclear cells of patients with ELANE exon 3 point mutations (Q97P and I118N), we designed experiments to recapitulate G-CSF signaling in SCN myelopoiesis. The patient-derived iPSC lines were characteristically similar to human embryonic stem cell lines and normal blood-derived iPSC lines with expression of SSEA-4, Tra-1-60, Tra-1-81, and CD9 pluripotency markers >85% in all lines as determined by FACS analysis at passage 10-15. All iPSC retained a normal karyotype and ELANE locus mutations of the original specimens. Both SCN iPSC lines and control iPSC lines (normal and immune-mediated congenital neutropenia) show functional G-CSF (50 ng/mL)-induced differentiation into the myeloid lineage. However, G-CSF failed to induce terminal granulocytic differentiation of SCN iPSC derived myeloid progenitors resulting in differentiation arrest at the promyelocyte/myelocyte stage. The hematopoietic cells derived from SCN iPSC line show reduced frequency of CFU-G and CFU-GM (CFU-G+CFU-GM, 22±1.15 in 10⁴ embryoid body cells from non SCN iPSC vs 4±2 in 10⁴ embryoid body cells from ELANE mutated SCN iPSC). Moreover, SCN iPSC promyelocytes show significantly increased apoptosis over controls as determined by Annexin-V analysis (31% Annexin-V+ in SCN iPSC derived promyelocyte vs 3% in control iPSC). Surviving SCN promyelocytes showed decreased reactive oxygen species activity and no phagocytic activity. In contrast, suprapharmacological doses of G-CSF (1000 ng/mL) rescued SCN iPSC-derived differentiation. It has been shown that NE, expressed in promyelocytes, can cleave recombinant G-CSF and CSF3R. We evaluated the possibility that inadequate G-CSF signaling in SCN iPSC was due to abnormal elastase activity. To this end, we supplemented 50 ng/mL G-CSF cultures with a specific, cell-permeant neutrophil elastase inhibitor (Sivelastat, 230 nM) to find rescued granulocyte differentiation arrest (31% neutrophil in myeloid differentiation culture of SCN iPSC derived hematopoietic progenitors in presence of Sivelastat vs 2% mature neutrophils without Sivelastat). In conclusion, SCN modeling through patient iPSC derived myelopoiesis and G-CSF driven granulocytic recapitulation unveil a mechanism of resistance to G-CSF therapy with important translational implications for therapy. Combination of low-dose G-CSF and NE inhibitory therapy may result in an efficacious, safer approach in SCN therapy.