부산대학교 도서관

Neutrophils are a type of immune cell that are difficult to study due to their short lifespan. To circumvent this, we developed an optimal culture protocol for the ex vivo production of neutrophils from peripheral blood CD34+ stem cells. Functional characterization of these cultured neutrophils revealed that they are morphologically similar to primary neutrophils, they have a similar proteome as primary neutrophils, and recapitulate most neutrophil antimicrobial activities, including a respiratory burst, phagocytosis, degranulation, cytokine release and bacteria killing. This makes cultured neutrophils an attractive model system to study neutrophil biology in health and disease. However, cultured neutrophils were shown to lack mature neutrophil surface markers, require stronger stimulation to induce neutrophil extracellular trap formation, and did not clear C. Albicans efficiently. Cultured neutrophils also possess an altered granule content and a higher rate of metabolic activity than primary neutrophils. This suggests that cultured neutrophils are in an immature state, making this approach an excellent system to study immature neutrophils. Subsequent optimisation of a scaled-down culture allowed for successful production of cultured neutrophils from small volumes of blood samples, allowing ex vivo culture of rare patient samples. This approach was used to examine Barth Syndrome. Morphological analysis, surface marker expression and neutrophil functional assays demonstrated that Barth CD34+ stem cells differentiate down the neutrophil lineage, suggesting that a proposed block in granulopoiesis does not underpin neutropenia in Barth Syndrome. Finally, a tool kit to manipulate protein expression in cultured neutrophils was established. We confirm that lentivirus can be used to express open reading frames, while an RNP/Cas9-gRNA approach can be used to efficiently knock out target proteins in cultured neutrophils without affecting in vitro differentiation. This was used to examine the role CD11b plays in neutrophil response to C. Albicans and begin to explore the function Ataxia telangiectasia mutated (ATM) protein plays in neutrophil antibacterial activity. This genetic tool kit establishes a model system which can now be applied to address a range of outstanding questions in the neutrophil field.