Hematopoietic colony-forming assay was designed to assess the hematopoietic potential and was performed according to the protocols described by Nakano et al. (2013 (link)). Briefly, the embryonic tissues including YS, caudal half, head, and heart regions were dissected and precultured on OP9 stromal cells for 4 d at 37°C in 24-well plates containing 1 ml α-MEM (Gibco) containing 20% fetal bovine serum (Gibco) and 1% penicillin/streptomycin supplemented with stem cell factor (50 ng/ml), thrombopoietin (5 ng/ml), IL-3 (IL-3, 5 ng/ml), IL-6 (IL-6, 5 ng/ml), and Flt-3 ligand (Flt-3L, 10 ng/ml). After 4 d, the tissues were transferred from 24-well plates into 1.5 ml tubes and dissociated mechanically by pipetting, and the stromal cells were removed through filtering. The cells were then transferred into methylcellulose supplemented with IL-3, IL-6, stem cell factor, and erythropoietin (MethoCult GF M3434; Stem Cell Technologies) in 1:10 ratio, followed by mixing well and plating with 400 μl medium at the bottom of the 24-well plates. The cells were cultured at 37°C for 10 d. The hematopoietic colonies were analyzed with microscope (ECHO/RVL-100-G) according to the manufacturer’s instruction.