Sup Video 1. Time lapse live cell imaging of hPSCs blood differentiation from day 4 to day 17

During the first wave of blood formation, non-adherent haematopoietic cells were shed from the adherent layer starting at day 4 forming haematopoietic colonies over the next 3 days (see 96-144 hours). Non-adherent cells were primary erythrocytes (Glycophorin A+).

Diffuse expression of SOX17-mCHERRY in the adherent layer appeared at 110 hours (day 4), but no RUNX1C-GFP cells at this stage.

SOX17-mCHERRY adherent cells condensed to form a reticular network from day 9-11 (192-264 hours). CD235a (GYPA) was used to distinguish primitive from definitive human haematopoiesis. Non-adherent cells did not express glycophorin A, but CD43, found on EMP cells at day 10 suggesting that the second wave of haematopoiesis had commenced.
On day 12 (300 hours) SOX17-mCHERRY+ clusters formed spindle-shaped structures with an intense fluorescent signal.

The first migratory RUNX1C-GFP+ cell appeared at day 14 (345 hours) following the formation of SOX17-mCHERRY arterial-like structures (third wave of haematopoiesis). The numbers of RUNX1C-GFP+ cells increased up to the end of the movie (day 17).

Some of the RUNX1C-GFP+ cells were associated with SOX17+ vasculature, though cells expressing both markers (RUNX1C-GFP+/SOX17+) were not observed by fluorescence microscopy.