Modeling Neutropenia by Utilizing Patient Derived Induced Pluripotent Stem Cells.

Congenital neutropenia is characterized by a reduced neutrophil count, decreased innate immunity and increased susceptibility to recurrent infections. While congenital neutropenia has various genetic causes, recent studies have linked mutations to this condition. , a key component of the vacuolar ATPase (V-ATPase) complex, is essential for osteoclast function, but its role in hematopoiesis remains unclear. We previously identified heterozygous mutations, including R736S, R736C, R736P, and E722D, in individuals with congenital neutropenia. However, the mechanism by which these mutations lead to impaired granulopoiesis remains unknown. To investigate the functional consequences of mutations, we generated induced pluripotent stem cells (iPSCs) from affected individuals and healthy controls. Using differentiation protocols, we assessed hematopoietic progenitor formation, proliferation, survival, and neutrophil differentiation. We observed significant defects in myeloid differentiation and increased cell death in patient-derived iPSC lines. CRISPR/Cas9-mediated correction of the R736C mutation restored normal neutrophil differentiation, confirming its pathogenic role. Immunofluorescence analysis revealed reduced expression and altered intracellular localization of the protein, characterized by a more diffuse cytosolic distribution in the mutant cell lines. Our findings suggest that mutations impair neutrophil development, likely through structural and functional disruption of the V-ATPase complex. This study provides new insights into the molecular basis of -associated neutropenia and highlights potential avenues for therapeutic intervention.