PS1232 RAPID MAST CELL GENERATION FROM GATA2 REPORTER HUMAN PLURIPOTENT STEM CELLS

Background: Mast cells (MC) are tissue resident immune cells with a key role in inflammation and defense against pathogens. MC dysfunction is involved in a broad range of common severe conditions such as allergies, autoimmune diseases, asthma or mastocytosis. Treatments to counteract the damaging effects of MC are limited and the development of new drugs is hampered due to the difficulty in obtaining these cells for research. We have developed a novel method for rapid production of MC using mouse and human pluripotent stem cells engineered with a Gata2 Venus (G2 V) reporter gene. Aims: To optimize the MC production from mouse and human embryonic/pluripotent stem cells (ESC/iPSC) in order to provide an efficient source of these cells for biomedical research and drug screening. Methods: Mouse G2 V cell line was previously generated by inserting an IRES‐Venus sequence into the genomic locus of Gata2 . Human ESC/iPSCs were targeted using CRISPR‐Cas9 to generate hG2 V reporter lines. In both cases, Venus fluorescent protein allowed us to isolate live cells expressing endogenous levels of Gata2 . G2 V ESC/iPSC were cultured in a 3‐step protocol: Cells were first differentiated into embryoid bodies (step1) for hematopoietic commitment (10 days for mouse and 8 days for human). Subsequently, Venus+ hematopoietic progenitors were FACS sorted and seeded on a monolayer of OP9 or H92–1 stromal cells for 5 days (step 2). Finally, V+ cells were sorted and plated in methylcellulose for MC expansion (step 3). After this step, cells were harvested and stained for MC markers for FACS analysis or treated for functional assay. The functionality of the obtained MC was assessed by measuring the levels of β‐hexosaminidase release after IgE stimulation. Results: With our 3 step protocol we were able to rapidly produce MC from mESC. After co‐culture, all Venus+ cells express leukocyte marker CD45 and 100% of Venus+CD45+ cells are ckit+ (MC progenitors) or ckit+FcεRα+ (MC). In order to test the functionality of the cells obtained, a FcεR1α‐mediated degranulation assay was performed. IgE‐sensitized G2V ‐mESC‐derived MC responded to antigen stimulation (measured by the percentage of β‐hexosaminidase release) similarly to primary mouse peritoneal MC used as control. The same G2 V reporter approach combined with our 3 step protocol allowed us to produce human phenotypic MC. After 5 days of OP9 co‐culture, 60% of V+CD45+ cells expressed cKit in their surface and 14% were cKit+FcεR1α+. We are currently optimizing our method to increase the numbers of human functional MC obtained. For this purpose, we are addressing the impact of different cytokines proven involved in MC development in our culture system. In addition, we are implementing the co‐culture with a human placental stroma cell line (H92–1) in order to give further support to our human ESC/iPSC‐derived MC. Summary/Conclusion: G2 V marks highly proliferative MC precursors and MC that appear in culture after 2 weeks of differentiation. The G2V ‐mESC‐derived MC cells are functional and respond to activation similarly to primary mouse MC. With our system, phenotypic human MC can be generated from human G2V‐ESC/iPSC reporter lines at least 6 times faster than current protocols. This method provides a rapid and readily accessible source of MC for biomedial research and drug screening.