Production of erythroid cells from human iPSC. (A) Schematic representation of the successive culture steps for production of cultured RBCs (cRBCs) from fibroblasts. Fibroblasts were transformed to iPSCs using the Yamanaka factors. Clumps of undifferentiated iPSCs were cultured in erythroid body (EB) medium for 28 d in the presence of a mixture of cytokines (cytokine profile 1). After 1 wk of culturing in normoxia, erythroid bodies were allowed to differentiate for 3 wk in normoxia/hypoxia, and the populations were analyzed by flow cytometry at day 28 or were allowed to mature further under cytokine profile 2 by sequential sets of cocktails and analyzed at day 58. Bars: (fibroblasts) 200 µm; (iPSC) 200 µm; (EB) 200 µm; (red blood cell, right) 2 µm. (B) CD analysis of various CD markers (CD34, CD45, CD36, CD71, and CD235a) under normoxia for sea-level samples. Each experiment was done in three replicates, and the experiment was done at least three times. The cells are cultured as erythroid bodies in cytokine profile 1 and as single cells in cytokine profile 2 (sequential mixture of cytokines). Error bars represent the mean ± SEM of at least two to three measurements. The experiment was repeated at least three times.