Differentiation of defined numbers of hESCs as spin EBs. (A) Schematic diagram of the spin EB method for hESC differentiation. (B-C) Immunofluorescence images of 3000 ENVY hESCs deposited in (B) flat- and (C) round-bottomed wells immediately after centrifugation. Aggregation of ES cells was only induced in the round-bottomed wells. Original magnification, 50×. Objective used was 5 ×/0.12 NA. (D) Percentages of wells containing myeloid or erythroid cells graphed as a function of the input number of cells in each well. Seventy-two wells were assayed at each point. (E) Sequential expression of differentiation stage–related genes in EBs harvested after the indicated number of days of differentiation. C indicates no template control. (F-K) Hematopoietic cells derived from spin EBs. (F-G) Bright-field microscopy of EBs differentiated for 11 days in suspension culture and then plated down for a further 14 days, revealing large numbers of (F) nonhemoglobinized and (G) hemoglobinized blood cells. Original magnification, 100 ×. Objective used was 10 ×/0.30 NA. (H-I) May-Grünwald-Giemsa–stained cytocentrifuge preparations after 28 days of differentiation showing neutrophils (n), macrophages (m), and mast cells (mc) in wells containing (H) myeloid cells and large numbers of maturing erythroid cells (ery) in wells containing (I) overtly hemoglobinized cells. Some cells with condensed chromatin apparently undergoing enucleation were observed (inset, arrow). Original magnification, 400 ×. Objective used was 40 ×/1.30 NA. (J,K) Flow cytometry of EBs dissociated (J) at day 11 and stained for expression of CD34 and CD38 and (K) at day 26 and stained for expression of Tie-2 and CD45. Percentages of cells within the indicated quadrants or regions are shown. All images were captured with a Zeiss Axiocam mounted on an Axiovert 200 inverted microscope using Axiovision software (Carl Zeiss, Göttingen, Germany). Image montages were created in Adobe Photoshop (Adobe Systems, San Jose, CA).