Figure 1. Study design.

(A) iHOXB4 ESCs were differentiated into EBs for 6 days in the presence of Dox. (B) Dissociated EB cells were analyzed and sorted by flow cytometry (FC). EB cells or their subpopulations were co-cultured with OP9 cells for 4 days in the presence or absence of Dox. (C) Co-cultured cells were analyzed and GFP⁺ cells were sorted by FC when HOXB4 expression was turned on. Regardless of HOXB4 status in co-cultures, FC sorting was performed on co-cultured cells based on forward and side scatters and on surface markers. Sorted cells were subjected to long-term reconstitution assays. The sorting process turned out to be useful for cell counts, removal of dead cells, and elimination of the remaining Dox.