Hematopoietic cell differentiation was performed as previously described (Ronn et al., 2015, Woods et al., 2011). Micrographs of cells from the cultures were taken at day 22 prior to whole well harvest. Floating and individualized cells were pooled, washed, and divided into two samples. One sample was used for assessment of hematopoietic lineage markers using FACS. The other sample was plated into methylcellulose (MethoCult H4435, StemCell Technologies) for colony-forming unit assay. The cells for FACS analysis were stained for mouse anti-human CD45/CD43/CD34/CD71 antibodies (BD Pharmingen; CD71APC, cat. no. 551374, clone M-A712. CD45 FITC or PE cat. no. 555482, 555483, clone HI30. CD43FITC cat. no. 555475, clone 1G10. CD34APC cat. no. 555824, clone 581), CD33/CD235a (GPA) antibodies (eBioscience, CD235a(Gly-A) cat. no. 12-9987-82, clone HIR2(GA-R2). CD33PE-Cy7 cat. no. 25-0338-42 clone WM-53(WM53)) and analyzed by a FACSCanto II flow cytometer.
