Clonogenic Assay of hPSC-derived HPCs

hPSC-derived HPCs were plated at 5.0 × 10² cells/0.5 mL in Methocult H4434 (StemCell Technologies, Vancouver, BC, Canada) to assess clonogenic colony-forming unit (CFU) capacity, as previously describe [25 (link)]. Cells were incubated at 37 °C for 14 days and manually scored. Each CFU well represents an independent biological assay, as input cells and MethoCult formulations were individually prepared for testing in single wells. CFU were stained with calcein green (Invitrogen, Waltham, MA, USA) in Hank’s Buffered Salt Solution (HBSS) for 30 min and imaged with the Operetta High Content Imaging System (PerkinElmer, Guelph, ON, Canada).