Figure 1. Erythroid cells differentiated from human embryonic stem cell sacs (ES sacs).

(A). Schematic diagrams of the procedure to establish human erythroid cells through human ES sacs. We generated ES sacs from embryonic stem (ES) cells (1×10e5) using Iscove’s Modified Dulbecco’s Medium (IMDM) containing vascular endothelial growth factor (VEGF) on irradiated C3H10T1/2 feeder cells for 15 days, as previously described [25]. We harvested spherical cells that emerged within the ES sacs, and at 2 days after culture of the spherical cells on irradiated OP9 feeder cells, the suspension cells were collected and differentiated into erythroid cells using stem cell factor (SCF) and erythropoietin (EPO) on OP9 feeder cells for 5 days, and EPO alone for the following 8 days. (B). After 15 days of culture, sac-like structures were generated from ES cells (Left image: 40×) and the ES sacs contained spherical cells (right image: 100×). (C). CD31+CD34+ hematopoietic endothelial cells comprised 22.2 ± 3.3 % of the spherical cells that were harvested from the ES sacs on day 15 (n=6), consistent with a hemangioblast-like phenotype. (D). After 13 days of culture for erythroid differentiation using the spherical cells (day 30), we observed small eosinophilic cells with high density chromatin by Wright-Giemsa staining consistent with erythroblasts. (E). We observed a high percentage of glycophorin A (GPA)+ in the differentiated erythroid cells (n=8). FL, fms-related tyrosine kinase 3 ligand; TPO, thrombopoietin; IL3, interleukin-3; BMP4, bone morphogenetic protein 4.