(19a) Human Stem Cell Derived Neutrophils As a Primary Neutrophil Model

As the first responders and the most abundant innate immune cell, neutrophils are a crucial part of the immune system. During an immune response, neutrophils migrate to sites of infection and eliminate bacteria through a series of anti-microbial functions including phagocytosis, production of reactive oxygen species, and NETosis. Improper regulation of neutrophil behavior contributes to many human diseases. Improper neutrophil function leads to chronic infections when neutrophils do not arrive at a site of infection or chronic inflammation when neutrophils persist at a site of infection. Current models for evaluating neutrophil behavior in vitro include cell lines and primary human neutrophils. Unfortunately, cell lines do not always accurately model many important neutrophil functions such as NETosis and phagocytosis and primary human neutrophils are hard to genetically manipulate. Recently, stem cell derived neutrophils have emerged as a possible model for primary neutrophils that would allow for genetic manipulation and the modeling of human disease.

In order to properly model primary human neutrophils, hiPSC-derived neutrophils must display the same morphology, migration, and anti-microbial behavior as primary human neutrophils. We have found that the morphology of hiPSC-derived neutrophils is indistinguishable from primary human neutrophils. Furthermore, hiPSC-derived neutrophils have the characteristic tri-lobed nucleus common to primary neutrophils. In order to reach sites of inflammation neutrophils must be capable of migrating to known neutrophil chemoattractants. We studied neutrophil chemotaxis using a previously published microfluidic device that creates a stable chemokine gradient and found that hiPSC-derived neutrophils efficiently chemotax to a source of the bacterial peptide fMLP, a common neutrophil chemoattractant. Finally, hiPSC-derived neutrophils must be able to perform anti-microbial functions. We visualized hiPSC-derived neutrophils in co-culture with pseudomonas aeruginosa using time-lapse microscopy. In contrast to popular neutrophil cell lines that lack the ability to phagocytose bacteria, we found that hiPSC-derived neutrophils were capable of efficiently phagocytosing pseudomonas over an extended period of time. We have also found that, upon activation, hiPSC-derived neutrophils produce neutrophil extracellular traps, proving these cells are capable of performing several anti-microbial functions.

Overall, we have found that hiPSC-derived neutrophils accurately recapitulate many characteristics and can serve as an excellent model of primary human neutrophils. We have shown that hiPSC-derived neutrophils share the morphology, migration capabilities, and anti-microbial functions of primary human neutrophils. hiPSC-derived neutrophils have the characteristic tri-lobed nuclei, are capable of chemotaxis to fMLP, a physiologically relevant chemokine, and poses the ability to phagocytose bacteria. In the future, hiPSC-derived neutrophils as a model will allow for more in-depth study of primary human neutrophil function.