FACS-sorted MniPSC-CD34⁺ cells give rise to CD45⁺CD34⁻ hematopoietic progeny. (A) Schematic of differentiation of MniPSC lines 3 and 7. Both MniPSC lines were induced days 0 to 8 with the optimized protocol outlined in Figure 2 with 2μM PGE2 added to EBs on days 1-8 of differentiation. On day 8, EBs were dissociated into single cells, immunostained with PE-conjugated anti-CD34 antibody (clone 563), and sorted by FACS for the CD34^high fraction. CD34^high cells were plated in cStemPro plus cytokines (50 000 CD34^high cells per well of a 6-well plate) on irradiated OP9 stromal cells. (B) Percentages of CD34^high cells that coexpress hematoendothelial markers on the indicated days of differentiation, which include presorted (day 8) and postsorted populations (days 14, 21). (C) Absolute number of fluorphore⁺ cells for MniPSC lines 3 and 7 that were induced toward hematopoietic differentiation, sorted for the CD34^high fraction on day 8, and plated in OP9 coculture. Absolute cell yields corresponding to the indicated hematoendothelial subsets were calculated (absolute positive cell yield = 100 × [% fluorphore⁺ × viable cell yield]). Each number shown is ×10⁶ and is representative of the total fluorphore⁺ cell yield obtained from an input undifferentiated MniPS cell number of 1 million. (D) Comparison of hematopoietic colony-forming potential (CFUs) of unsorted and CD34-enriched cells from day 8 MniPSC line 3 and line 7 HPCs and pigtail macaque mobilized bone marrow. To evaluate MniPSC-derived HPCs, EBs were dissociated on day 8, kept in bulk (unsorted), or sorted by FACS and then plated in Methocult H4435. Error bars indicate SD of mean of 3 separate experiments. (E) Representative flow cytometry dot plots of MniPSC-derived HPCs on day 18 of differentiation. (F) Representative images of MniPSC Line 7-derived CD34^high cell-derived hematopoietic zones on OP9 stromal cells on day 21 of differentiation. Similar results were obtained from MniPSC line 3 CD34^high cells. Colonies were imaged on a Nikon Eclipse Ti-M (TiSR) microscope and photographed with a Nikon camera, in cStemPro media (10× [left] and 20× [right] objectives). Scale bar = 100 μm.