The three MEDEP lines exhibited differential expression of TER119 (a cell surface antigen specific for mature erythroid cells) and CD71 (transferrin receptor) (Figure 2A and Figure S4). For example, the expression of CD71 was slightly higher in MEDEP- E14 cells than in MEDEP-BRC5 cells (Figure 2A). TER1192CD712 cells differentiate first to TER1192CD71+ cells, subsequently to TER119++CD71+ cells, and then finally to TER119+CD712 cells [25]. Consistent with the differences in their cytokine dependency (Figure 1B), the MEDEPs appeared to represent different stages of erythroid differentiation. Despite these differences, following induction of differentiation in vitro by the methods described above, the expression of TER119 and CD71 in each of the MEDEP lines exhibited a pattern consistent with a more mature lineage (Figure 2A and Figure S4), indicating that each of the three lines was able to differentiate into a more mature lineage. At present, the cause of the variability between MEDEPs remains uncertain. However, these results clearly demonstrated that erythroid progenitor cells could be immortalized at different stages of their differentiation. Notably, the vast majority of cells in each MEDEP line could differentiate into more mature cells, although each MEDEP line included cells possessing abnormal karyotypes (Figure S5). This result strongly suggested that the cells possessing abnormal karyotypes still retained the potential to differentiate into more mature erythroid cells. In general, most immortalized cell lines are not necessarily homogenous in karyotype and/or characteristic, even after cloning. The emergence of cells possessing different karyotypes and/or different characteristics is often observed following long term utilization of immortalized cell lines. Hence, periodical recloning and selection of cell lines is recommended to maintain their characteristics. Following induction of differentiation in vitro, cell pellets appeared red while the cell pellets before differentiation appeared white (Figure 2B). In addition, the appearance of enucleated cells following differentiation was demonstrated by flow cytometric analysis using SYTO85 staining (Figure S6). Moreover, it was confirmed by morphological analysis that enucleated RBCs in addition to very mature erythroblasts were present following induction of differentiation (Figure 2C, arrows). In vivo proliferation and differentiation of MEDEP To evaluate the functional potential of MEDEP cells in vivo, we established a subline of MEDEP-E14 expressing Venus [26] as a marker, MEDEP-E14-Venus. Although the expression of TER119 was slightly higher in MEDEP-E14-Venus cells than in MEDEP- E14 cells (compare Figure 2A and Figure 3A), MEDEP-E14- Venus cells retained the ability to proliferate (data not shown) and differentiate into more mature erythroid cells in vitro (Figure 3A). In general, the ablation of endogenous hematopoietic cells in mice is required to detect transplanted hematopoietic cells efficiently. Acute anemia induced by phlebotomy or hemolysis is commonly applied in the study of urgent erythropoiesis [25,27]. Hence, we induced acute anemia in mice by intraperitoneal injection of phenylhydrazine, an inducer of hemolysis, and transplanted MEDEP-E14-Venus cells (26107 cells/mouse) 24 hours later. Three days after cell transplantation, Venus- positive cells were present in the bone marrow and spleen (Figure 3B). Since the spleen is the major organ supporting urgent erythropoiesis [25], the transplanted cells were observed more abundantly in the spleen than in the bone marrow (Figure 3B). Venus positive cells (the transplanted cells) demonstrated a phenotype consistent with differentiation into more mature erythroid cells (Figure 3B) compared to the phenotype of the cells just before transplantation (Figure 3A, before differentiation). Of ESC-Derived Erythroid Cells note, MEDEP-E14-Venus cells differentiated into much more mature lineages in vivo than they did in vitro (compare Figure 3A and Figure 3B). To evaluate whether transplanted cells can proliferate in vivo, we determined the proportion (%) of Venus-positive cells and calculated the absolute number of Venus-positive cells in the spleen in a cell transplantation experiment performed similarly to that shown in Figure 3B. The absolute number of Venus-positive cells was elevated approximately two fold at three days after cell transplantation compared to that at one day (Figure 3C). This result indicates that transplanted cells can proliferate in vivo. However, this proliferation was transient and the transplanted cells did not form a tumor in vivo, as shown below. The expression of Venus in the transplanted cells decreased following the differentiation of them, i.e., the expression of Venus was lower in TER119++ cells than in TER119+ cells (Figure 3A). Thus, although we could not detect Venus-positive cells in peripheral blood (data not shown), it was highly likely due to the disappearance of Venus following the terminal differentiation. To confirm that MEDEP could differentiate into terminally-differentiated RBCs in vivo, we performed the following experiments. Increase of RBC number in mice suffering from acute anemia following transplantation of MEDEP MEDEP cells (26107 cells/mouse) were transplanted 24 hours after induction of acute anemia. As a control experiment, MEDMC cells (26107 cells/mouse) were transplanted into control mice. Since 26107 transplanted RBCs correspond to a mere 2 ml of transfused cells, the number of RBCs in transplanted mice will only increase if these transplanted MEDEP cells proliferate to some degree and differentiate into terminally-differentiated RBCs in vivo. Five days after the transplantation, the peripheral blood was subjected to a blood count. The transplantation of MEDEP- E14 significantly ameliorated anemia compared to the control (Figure 4A). The data obtained from the mice transplanted with control cells (Figure 4A) did not differ significantly from the data obtained from the anemic mice that were not transplanted with any cells (data not shown). Since the RBC count in peripheral blood reflects the number of enucleated cells, whereas erythroblasts (nucleated cells) are counted as white blood cell (WBC), the increased number of RBC observed in mice transplanted with MEDEP cells (Figure 4A) indicated that the transplanted MEDEP cells could differentiate into enucleated cells very efficiently. Since the life span of RBCs is approximately 50 days in the mouse, it is highly likely that the RBCs produced from the transplanted MEDEP cells accumulated in the transplanted mouse. Increases in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and MCH concentration (MCHC) (Figure S7) are commonly observed in the recovery phase of acute anemia [25]. In addition, an increase in the number of WBC observed in the recovery phase of acute anemia (Figure S7) is due to the presence of erythroblasts in the peripheral blood, since erythroblasts are counted as WBC by the automatic counter [25]. Given that there was no difference in MCV, MCH and MCHC levels between the two groups in the recovery phase of acute anemia (Figure S7), RBCs derived from MEDEP cells in vivo appeared to possess character- istics quite similar to those derived from erythroid progenitor cells in the host mice. Twenty-six days after transplantation (27 days after the induction of acute anemia), all mice had recovered from the anemia and there were no differences in the blood counts of the two groups (Figure 4A and Figure S7). PLoS ONE | www.plosone.org 5 February 2008 | Issue 2 | e1544