From Marrow to Model: a human bone marrow-derived neutrophil assay to study neutrophil development and function 2257049

Introduction Neutrophils play critical roles in innate immunity, autoimmune disease and cancer, however drug development to target these cells have been impeded by poor in vitro systems. There is a need to develop reliable long-term culture methodology to generate abundant neutrophils at various developmental stages, not possible with peripheral blood-derived neutrophils, the current neutrophil biology gold standard. Methods Human bone marrow isolated from healthy volunteers was enriched for CD34+ hematopoietic stem cells, then differentiated in vitro to neutrophils for up to 23 days. Cells from various developmental stages were assessed for morphologic changes, cell surface maturation markers and functional responses. Results Bone marrow differentiated neutrophils (BMN) exhibited nuclear lobulation by day 8 and maturation markers by day 13. Cell surface marker expression patterns on BMN and morphology matched those of peripheral blood neutrophils (PBN). Additionally, BMN exhibited full functional capabilities like PBN including elevated ROS and NET production. Moreover, BMN is also capable of phagocytosis. Conclusion In vitro culture of CD34+ cells from human bone marrow can give rise to large numbers of mature neutrophils that are morphologically and functionally like PBN, but unlike PBNs, can survive for 3 weeks in culture. This method can provide better understanding of neutrophil development and function and support drug development in neutrophil mediated diseases.