Production of enucleated RBCs from hESCs. (A) Protocol to produce RBCs from hESCs. Embryonic stem (ES) cells were cocultured with irradiated FH-B-hTERT cells for 14 to 35 days. The cells were then dissociated and CD34⁺ cells were sorted and seeded in liquid culture in the presence of Flt-3L, stem cell factor (SCF), erythropoietin (EPO), bone morphogenetic protein (BMP)-4, and interleukin (IL)-3 (amplification 1, 7 days) and then of insulin-like growth factor (IGF-)1, SCF, EPO, BMP-4, and IL3 (amplification 2, 7 days). Terminal maturation was induced by culture on an MS-5 feeder layer for 10 days (first 3 days in the presence of EPO, then without any cytokines). (B) Proliferation rate of CD34⁺ cells from fetal liver (FL), cord blood (CB), and peripheral blood (PB; first panel) and of hESC-derived CD34⁺ cells from 14-, 21-, and 35-day coculture (panels 2-4) placed in condition described in panel A. The red curves represent the actual amplification. The dotted lines represent the calculated amplification assuming 1% survival of the seeded cells (see “Lengthening the coculture time leads to the production of red blood cells that can enucleate”). (C) Morphologic characterization and enumeration of the erythroblasts demonstrated that differentiation was relatively synchronous during the liquid culture period. (D) Flow cytometric analysis with anti-CD 235a (glycophorin A) antibodies of erythrocytes obtained after 14 or 35 days of coculture and 24 days of liquid culture. The bar graph summarizes the results of 3 experiments. (E) Micrographs of red cells obtained at the end of step 4 of our culture system. The total length of the 4-step culture varied between 24 and 29 days because steps 1 and 2 were occasionally lengthened for practical reasons (see “Amplification and differentiation of CD34⁺ cells”). Fourteen-day cocultures yielded large nucleated RBCs similar to cells produced in the yolk sac. Thirty-five-day cocultures yielded orthochromatic and enucleated RBCs similar to cells produced in the FL. (F) Size of the erythroblasts produced in culture. Sizes were estimated microscopically after 21 or 24 days of liquid culture. Poly-E, polychromatic erythroblasts; Ortho-E, orthochromatic erythroblasts; RBC, enucleated red blood cells. The cells from 35-day culture were closer in size to FL-derived cells than to the 14-day cells. (G) Bar graph summarizing globin expression (determined by HPLC) in erythroblasts produced either in culture from FL- or PB-derived CD34⁺ cells, or circulating at the time of harvest of the CD34⁺ cells. Globin levels observed in vitro closely matched the levels observed in vivo. Black bars, expression in in vitro–produced cells; white bars, expression in in vivo–produced cells. Percent α-globin was calculated as 100 × α-globin/(α-globin + ζ-globin). Percent γ-globin was calculated as 100 × γ-globin/(γ-globin + β-globin). Error bars represent SD.