Effective and Rapid Generation of Functional Neutrophils from Induced Pluripotent Stem Cells Using ETV2-Modified mRNA

Abstract

Summary: Human induced pluripotent stem cells (hiPSCs) can serve as a versatile and scalable source of neutrophils for biomedical research and transfusion therapies. Here we describe a rapid efficient serum- and xenogen-free protocol for neutrophil generation, which is based on direct hematoendothelial programming of hiPSCs using ETV2-modified mRNA. Culture of ETV2-induced hematoendothelial progenitors in the presence of GM-CSF, FGF2, and UM171 led to continuous production of generous amounts of CD34+CD33+ myeloid progenitors which could be harvested every 8–10 days for up to 30 days of culture. Subsequently, myeloid progenitors were differentiated into neutrophils in the presence of G-CSF and the retinoic acid agonist Am580. Neutrophils obtained in these conditions displayed a typical somatic neutrophil morphology, produced reactive oxygen species, formed neutrophil extracellular traps and possessed phagocytic and chemotactic activities. Overall, this technology offers an opportunity to generate a significant number of neutrophils as soon as 14 days after initiation of differentiation. : In this article, Slukvin and colleagues describes a protocol for a rapid efficient feeder-, serum-, and xenogen-free protocol for neutrophil generation from hiPSCs, which is based on direct hematoendothelial programming of hiPSCs using ETV2-modified mRNA. Keywords: ETV2, hemogenic endothelium, neutrophils, human iPSCs, hematopoietic differentiation, modified mRNA, myeloid progenitors