Human mast cell differentiation optimization to study MRGPRX2-induced activation in vitro

Intro The mas-related G protein-coupled receptor member X2 (MRGPRX2) on mast cells (MCs) is suggested as new target for modulating asthma. A representative human MC model is needed to study IgE-independent activation via MRGPRX2. Methods Human MCs were cultured from progenitors of buffy coat and fresh blood samples (80 mL). CD34+ cells were cultured in a serum-free methylcellulose medium during 3 or 4 weeks with supplementation of different concentrations of IL-3 and/or IL-6, IL-9 and/or SCF, in presence or absence of FBS. Immunophenotyping of cells was performed by flow cytometric analysis of expression of CD45, CD117, FcεRI, and MRGPRX2. Degranulation upon activation was analysed by flow cytometric CD63 expression. Results CD34+ cells were differentiated (Cop N, et al. 2017) into viable MCs expressing both FcεRI and CD117 (46-89% of CD45+ cells). MRGPRX2 was expressed on 46% of MCs cultured in the presence of IL-3 (100 ng/mL during wk 1) and IL-6 (100 ng/mL during wk 1, 20 ng/mL during wk 2-4). Addition of IL-6 substantially increased MRGPRX2 expression compared to conditions with only IL-3 (30% of MCs). MRGPRX2 expression remained stable after adding IL-9 or further extending the protocol. Addition of FBS (10%) in wk 4, increased MC viability (45% to 62%) but dramatically decreased MRGPRX2 expression (46% to 3%). In order to study whether MRGPRX2 could be functionally activated, we stimulated cells with substance P (74 μM), resulting in increased CD63 expression. Conclusion This study demonstrates that adding FBS substantially decreased and IL-6 increased MRGPRX2 expression during in vitro differentiation, resulting in functional MCs, enabling research on MRGPRX2 dependent MC activation.