Optimal ex vivo production of functional neutrophils is dependent on the source of CD34+ human hematopoietic progenitors.

Abstract

Neutrophils serve as the first line of defense against invasive bacterial and fungal pathogens. The loss of circulating neutrophils leaves patients at a critical risk of life-threatening infections. In this study, we optimized conditions for expanding human precursor neutrophils ex vivo while preserving the functional capacity of mature neutrophils. We evaluated several CD34+ hematopoietic stem cells (HSCs) from various sources, including umbilical cord blood (UCB), adult bone marrow (BM), and cadaveric sources. UCB-derived CD34+ cells consistently demonstrated the highest expansion capacity, achieving an additional two cell divisions compared with BM-derived cells. Surface receptor profiling demonstrated that all sources resulted in mature neutrophil differentiation, although UCB-derived cell sources exhibited higher expression of maturation markers CD11b, CD15, and CD66b, in conditions expanded with the small molecule UM729. Functionally, neutrophils derived from all cell sources retained the ability to phagocytose and produce reactive oxygen species (ROS), with enhanced activity following antibody-dependent opsonization. To better understand the impact of opsonization, Fc receptor expression levels were assessed in addition to profiling changes in complement and adhesion receptor expression. Single-cell expression analysis confirmed that ex vivo differentiation was consistent with known patterns of myeloid differentiation, leading to distinct neutrophil subpopulations. Notably, mature neutrophils generated ex vivo were transcriptionally distinct from freshly isolated primary cells. Overall, our findings demonstrate that UCB-derived precursors offer the highest expansion potential for generating neutrophil precursors, able to mature into fully functional neutrophils. These results provide valuable insights into optimizing human neutrophil production as a promising cellular therapy for neutropenic individuals.