Isolation of Haemogenic Endothelium from Embryoid Bodies

Embryoid bodies were dissociated on day 8 by digestion with 0.05% trypsin for 5 min at 37 °C, pipetted thoroughly with p1000 to generate a single-cell suspension and washed with PBS + 2%FBS. Dissociated embryoid bodies were immediately processed for isolation of haemogenic endothelium. Cells were resuspended in 1 mL PBS⁺2%FBS and incubated with human CD34 MicroBead kit for 1 h (Miltenyl Biotec, 130-046-702). After incubation, cells were washed with PBS⁺2%FBS and isolated by magnetic cell isolation using LS columns (Miltenyl Biotec, 130-042-401). Sorted CD34⁺ cells were resuspended in supplemented StemPro-34 medium, containing Y-27632 (10 μM), TPO (30 ng ml⁻¹), IL-3 (10 ng ml⁻¹), SCF (50 ng ml⁻¹), IL-6 (10 ng ml⁻¹), IL-11 (5 ng ml⁻¹), IGF-1 (25 ng ml⁻¹), VEGF (5 ng ml⁻¹), bFGF (5 ng ml⁻¹), BMP4 (10 ng ml⁻¹), and FLT3 (10 ng ml⁻¹) as reported^{20 (link)} and seeded at a density of 25 × 10³ to 50 × 10³ cells per well onto thin-layer Matrigel-coated 24-well plates. All recombinant factors were human and most were purchased from Peprotech.