Enforced expression of RUNX1a in hESC (H9) and sequential hematopoietic differentiation through spin EBs. (A) Schematic representation of lentiviral constructs used to express RUNX1a. EGFP, enhanced green fluorescent protein; HA, human influenza hemagglutinin; MSCV, murine stem cell virus; PGK, phosphoglycerate kinase; PURO, puromycin. (B) Expression of transduced RUNX1a in hESC (H9) cells detected by RT-qPCR after lentiviral infection and puromycin selection. GAPDH is used as an internal control. Results from parental cells (WT) that represent endogenous RUNX1a are set as 1. Bar chart represents RUNX1a expression in vector or RUNX1a transduced cells relative to parental cells. Error bars represent SDs of 3 independent experiments. (C) Expression of transduced RUNX1a in hESC (H9) cells detected by western blot using anti-HA antibody after lentiviral infection and puromycin selection. α-Tubulin is used as a loading control. (D) Cell morphologies of sequential EB formation from H9 cells at day 5 and 11 under spin EB differentiation condition. Hemato-endothelial structure could be observed in RUNX1a transduced EBs since day 5 EBs. Scale bars, 50 μm. (E) Percentage of EBs containing typical sac (cystic) structure at spin EB day 7 and 11. Error bars represent SDs of 3 independent experiments. *P = .0158, **P = .0347.