ESC-Derived Erythroid Cells Figure 1. Characteristics of erythroid cell lines derived from mouse ES cells, MEDEP. (A) Morphology of two erythroid cell lines, MEDEP-E14 and MEDEP-BRC5. Wright-Giemsa staining. (B) Cytokine dependent proliferation. Cells (16105 cells/ml) were cultured in various conditions for three days. The added factor(s) is shown at the bottom. None, no specific factor. SCF, stem cell factor. EPO, erythropoietin. Broken line, the number of cells at the start of culture. Values are mean6S.D. Results shown are representative of several independent experiments performed at different time points after establishment of the cell lines. (C) RT-PCR analyses. Oct-3/4 and Nanog, transcription factors specific for ES cells. GATA-1 and EKLF (Erythroid Kru¨ ppel- like factor), transcription factors specific for erythroid cells. EPOR, erythropoietin receptor. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. NC, negative control without cDNA. Day 0, E14TG2a cells before differentiation. Day 4, 7, 10, 14 and 21, the cells following induction of differentiation into hematopoietic cells from E14TG2a by the method described in Table 1 (Method A). The cycle numbers performed in each PCR are shown at the right. Results shown are representative of two independent experiments. doi:10.1371/journal.pone.0001544.g001 has previously been reported [6], all MEDEP lines appeared to be derived from adult type erythroid progenitor cells. In vitro differentiation of MEDEP Next, we evaluated the potential of MEDEP cells to differentiate into more mature erythroid cells. We found that all MEDEP lines could differentiate into more mature erythroid cells by the following treat- ments: deprivation of EPO for MEDEP-E14 (Figure 2A); deprivation of SCF and addition of EPO for MEDEP-BRC5 (Figure 2A); and deprivation of SCF and dexamethasone and addition of EPO for MEDEP-BRC4 (Figure S4). EPO appeared to be necessary for MEDEP-BRC5 and MEDEP-BRC4 cells to maintain cell viability during the differentiation process (data not shown). PLoS ONE | www.plosone.org 3 February 2008 | Issue 2 | e1544