GATA1 knockout in human pluripotent stem cells generates enhanced neutrophils to investigate extracellular trap formation

Human pluripotent stem cell (hPSC)-derived tissues can be used to model diseases and validate targets in cell types that are challenging to harvest and study at-scale, such as neutrophils. Neutrophil dysregulation, specifically unbalanced neutrophil extracellular trap (NET) formation, plays a critical role in the prognosis and progression of multiple diseases, including COVID-19. hPSCs can provide a limitless supply of neutrophils (iNeutrophils) to study these processes and discover and validate targets in vitro. However, current iNeutrophil differentiation protocols are inefficient and generate heterogeneous cultures consisting of different granulocytes and precursors, which can confound the study of neutrophil biology. Here, we describe a method to dramatically improve iNeutrophils yield, purity, functionality, and maturity through the deletion of the transcription factor GATA1. GATA1 knockout (KO) iNeutrophils are nearly identical to primary neutrophils in cell surface marker expression, morphology, and host defense functions. Unlike wild type (WT) iNeutrophils, GATA1 KO iNeutrophils generate NETs in response to the physiologic stimulant lipopolysaccharide (LPS), suggesting they could be used as a more accurate model when performing small-molecule screens to find NET inhibitors. Furthermore, through CRSPR/Cas9 deletion of CYBB we demonstrate that GATA1 KO iNeutrophils are a powerful tool in quickly and definitively determining involvement of a given protein in NET formation.