Ssea 1

Eggs were collected shortly after fertilization from the poultry institute of the Chinese Academy of Agricultural Sciences Experimental Poultry Farm. A total of 18,340 eggs were collected and used for isolation of three different experimental groups: (1) ESCs, which were used immediately, and (2) PGCs, and (3) SSCs, which were both incubated at 37°C and 75% relative humidity for 5.5 and 18 days respectively, prior to use.
Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (U.S.A.). Mitomycin-C was obtained from Roche. β-Mercaptoethanol, chicken serum, L-glutamine, sodium pyruvate, trypsin, collagen enzyme I, human stem cell factor (HSCF), basic fibroblast growth factor (BFGF), human insulin-like growth factor (HIGF), and murine leukemia inhibitory factor (mUF) were acquired from Sigma–Aldrich. Antibodies specific to the following proteins were purchased: SSEA-1 (Biolegend, San Diego, CA, U.S.A.; dilution ratio 1:1000), Sox2 (abcam, Cambridge, England; dilution ratio 1:1000), SSEA1 (abcam, Cambridge, England; dilution ratio 1:1000), C-kit (SouthernBiotech, Birmingham, AL, U.S.A.; dilution ratio 1:1000), integrin α6 and integrin β1 (Millipore; dilution ratio 1:1000), and goat anti-mouse IgM (flourescein isothyocyanate [FITC] labeled; Bio-Synthesis, Inc., Texas, U.S.A.; dilution ratio 1:1000).

Tracheal epithelial cells were collected from the lungs of Cre-inducible reporter mice and plated on 4 μm pore transwell membranes (Corning). Cultures were grown in mTec⁺ for 2 days of proliferation then switched to mTec-serum free media for differentiation. After 21 days of differentiation, the pseudostratified cultures were infected with IAV_Cre then harvested at 1 dpi (to measure actively infected cells) and 8 dpi (to measure survivor cells after the clearance of viral infection). These cultures were then fixed with 2% PFA and antigen retrieval was performed in a citrate buffer. Samples were then stained with antibodies for active infection (HA, PY102), basal stem cells (Krt5, Biolegend, clone Poly19055, cat 905501), ciliated cells (FoxJ1, eBioscience, clone 2A5, cat 14–9965), secretory cells (SSEA-1, Biolegend, clone MC-480, cat 125611) using tdTomato as an endogenous marker of infection. Wholemount membranes were mounted in Prolong Diamond (Life Technologies) then imaged on a SP5 inverted confocal microscope (Leica) and images processed using Fiji.