ABSTRACT
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.
Subject(s)
COVID-19 , Chronobiology DisordersABSTRACT
BackgroundDeanery teaching for Greater Manchester and Lancashire (GML) Paediatric Trainees previously occurred monthly in Central Manchester. This was poorly attended as our deanery covers a wide geographical area so trainees did not have the option to drop by for part sessions if they were on call, those on nights and leave would miss it completely and rota pressures meant some trainees were left holding the fort on the ward resulting in missed and unequal learning opportunities. Covid brought further challenges of shielding trainees who could not safely attend and restrictions on face to face teaching.Our aim was to overcome these challenges by developing an online learning platform for GML paediatric trainees where deanery teaching can be accessed virtually and provide an online home for trainees to encourage engagement with other learning opportunities.ObjectivesDevelop a GML Paediatric website to act as an online home for traineesReduce barriers that prohibits attendance at deanery teachingBuild an archive of secure teaching videos for education that can be accessed remotely 24/7Provide flexibility so trainees can engage with learning around their home and work lifeImprove engagement with learning by promoting the website and educational opportunities on our new social media pageDeveloping a GML logo bringing together trainees all across our regionMethodsTrainee led committeeLaunch of an online advanced learning platform allowing recording capabilities and secure storage of deanery teaching allowing sessions to be watched live from home, work or stored to watch laterAcquired funding for recording equipmentDevelop a GML trainee website acting as an online home for trainees, promoting all regional education opportunitiesDevise a webinar series as a resource for trainees on core subjects and career adviceA diary of all study leave opportunities available to trainees on the website‘Communicating the vision’ to trainees via Twitter and a new online education newsletterResultsPre project only 13% of trainees attended deanery teachingPost project attendance at deanery teaching has increased by 96.6%There have been 317 visits to our new GML Paediatric website in 3 months- this is at least 3 views per dayIncreased social media presence directing trainees to visit the websiteConclusionsWe have demonstrated how a dedicated committee of trainees has significantly increased the number of attendees at deanery teaching and increased trainee engagement with learning opportunities through the development of an online learning platform despite being in the mists of a pandemic.