Apelin 36

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)).

After an overnight fast, the rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (300 mg/kg). A burr hole for ICV administration was carefully made in the skull at 0.8 mm dorsal and 1.6 mm lateral to the right from the bregma using a dremel drill. Total 10 µl apelin-13 (0.03 µg/µl), 10 µl apelin-36 (0.05 µg/µl) (Phoenix Pharmaceuticals, Belmont, CA, USA) (dissolved in aseptic PBS) or 10 µl vehicle only (PBS) were administered into the right lateral ventricle 2 h after MCAO, using a 10 µl microsyringe at the following stereotactic coordinates (AP: −0.8 mm; ML: 1.6 mm; DV: −3.8 mm) as we described previously (16 (link), 17 (link)). The experiments were not performed in a blinded manner.