Isolation and Differentiation of Intestinal Neurospheres

Neurosphere culture and differentiation was essentially carried out as described before^{39 (link)}. In brief, the intestine were dissected from embryos approximately 18 days p.c., minced and washed four times in HBSS/2% FBS (4 min, at 400g). Tissue was digested for 15 min at 37°C in HBSS supplemented with 0.05% Trypsin-EDTA solution (Gibco) and 50 μg/ml DNaseI. After digestion the solution was vortexed and filtered through a 70 μM cell strainer. Cells were plated on an ultralow adherent plate (Corning) in DMEM/F12 supplemented with N2 and Antibiotic-Antimycotic solution (all Gibco) and expanded for 4-5 days. EGF and FGF (20 ng/ml, both R&D) were added to the culture. For differentiation, cells were plated on a 96-well flat-bottom adherent plate (Corning) coated with fibronectin (20 μg/ml in PBS, Sigma) in Neurobasal medium supplemented with B27 and Antibiotic-Antimycotic solution (all Gibco) for 15 days. For co-culture, sort-purified ILC2s were added to the culture with IL-2 and IL-7 (20 ng/ml each).