BPS2026 – In vitro generated neutrophils reveal high deformability is an emergent property

Neutrophils must deform their nuclei up to <10% of the cross-sectional area to migrate in vivo, yet when this ability emerges remains unresolved. Cancer-derived models fail to reproduce the multilobulated nuclear morphology and envelope composition of primary neutrophils. Here, we developed a platform to differentiate neutrophils from CD34 + stem cells and investigate when high nuclear deformability emerges. We cultured CD34 + stem cells with growth factors and cytokines to differentiate them into functional neutrophils (dCD34-neutrophils) in 14 days with 76.5 ± 6.3-fold expansion. We showed that dCD34-neutrophils expressed canonical neutrophil markers (99% CD15 + , 78% CD11b + ), generated reactive oxygen species, and underwent NETosis comparable to primary human neutrophils. Using quantitative microscopy, we showed that nuclear circularity, an indicator of multilobulation, decreased from 0.84 to 0.77 as cells transition from progenitors (D3) to dCD34-neutrophils (D16). Coincidently, nuclear envelope composition changed: Lamin A/C and B1 decreased by 1.6- and 2.7-fold, respectively; Lamin B receptor (LBR) increased by 7.3-fold, inversely correlating with nuclear circularity. To determine if nuclear remodeling correlates with deformability, we measured migration speeds of progenitor and dCD34-neutrophils using microfabricated channels with and without constrictions. Migration speed increased from 7.7 ± 3.1 μm/min in progenitors to 14.1 ± 4.9 μm/min in late progenitors. In 2 μm constrictions, progenitors slowed (9.9 ± 3.9 before vs. 5.5 ± 2.6 inside), whereas dCD34-neutrophils maintained high speed (11.4 ± 6.2 before vs. 14.4 ± 7.6 inside). After constriction, progenitors remained slow (5.9 ± 2.6), while dCD34-neutrophils kept high speed (14.3 ± 6.2). Together, our data show that high nuclear deformability emerges late in neutrophil differentiation and correlates with LBR upregulation and Lamin A/C and B1 reduction. dCD34-neutrophils provide a platform to dissect how nuclear envelope composition regulates biophysical mechanisms of neutrophil functions in health and disease.