Quantitative Induction of Primitive Streak in hESCs

The capacity of BMP-4 to quantitatively promote primitive streak induction in human embryonic stem cells (hESC) was determined by using a MIXL1-GFP reporter cell line [14 (link)], as described previously [15 (link)]. In brief, single-cell suspensions were seeded in 12-well suspension plates (Greiner, Austria) at 0.33 × 10⁵ cells/ml in mTeSR medium (Stem Cell Technologies, Canada) supplemented with 10 μM Y-27632 inhibitor (kindly provided by Gerald Dräger, Leibniz University Hannover, Germany). Suspension aggregates formed within 24 h h. After 4 days, primitive streak induction was initiated by changing the medium to RPMI medium supplemented with 2% B27 supplement (Life Technologies, USA), 3 μM CHIR99021 inhibitor (kindly provided by Gerald Dräger, Leibniz University Hannover, Germany) and BMP-4 concentrations as indicated in the results section. Commercial rhBMP-4 (R&D Systems, USA) from NS0 mammalian cell culture was used as a positive control. Aggregates were harvested after 24 h and dissociated using Accutase for 5 min at 37 °C. The GFP signal was measured with a BD Accuri C6 flow cytometer (BD Biosciences, USA) and analyzed using FlowJo (v10; TreeStar, FlowJo LLC, USA).