Coaggregation of Embryonic Progenitor Cells

E9.5 and E11.5 embryos were dissected as described (Rybtsov et al., 2014 (link)). Tissues were incubated with collagenase/dispase solution (0.12 mg/mL) (Roche) at 37°C as described (Taoudi et al., 2008 (link)), washed, and resuspended in PBS (Sigma) containing 3% FCS and then dissociated by pipetting. After dissociation (and sorting) 1 e.e. of the specific cell populations (e.g., type I or type II pre-HSCs) were coaggregated with 10⁵ OP9 cells. Five to ten coaggregates (i.e., 5–10 e.e.) per experimental variant were cultured in Iscove's modified Dulbecco's medium (Invitrogen), with 20% of pre-selected, heat-inactivated FCS, L-glutamine, and penicillin/streptomycin supplemented with murine recombinant cytokines (SCF, IL3, and Flt3) each at 100 ng/mL (PeproTech) and various concentrations of APLNR ligand peptides, including APELIN 36 (LVQPRGSRNGPGPWQGGRRKFRRQRPRLSHKGPMPF), its cleaved bioactive pyroglutamyl form (Pyr1) APELIN 13 (QRPRLSHKGPMPF), and APELA 21 (LYRHACPRRRCIPLHSRVPFP) (Phoenix Pharmaceuticals). Coaggregates were cultured on floating 0.8 μm AAWP 25 mm nitrocellulose membranes (Millipore) for 6 days then dissociated using collagenase/dispase as described (Rybtsov et al., 2014 (link)).