ESC-Derived Erythroid Cells . ... Table 3. Blood count after induction of acute anemia and cell transplantation. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... RBC Hb Ht MCV MCH MCHC WBC Platelet . .. Experiment A . . .. Day 5 . ... MEDEP-E14-Venus (N = 3) 502.0630.4* 14.160.7* 36.761.0* 73.363.9 28.160.6 38.461.3 680.3686.1 97.1614.6 . ... MEDMC-NT2 (N = 3) 401.0611.4 10.860.3 29.260.8 72.860.6 26.960.2 37.060.5 783.3653.2 98.063.7 . .. Day 26 . .... MEDEP-E14-Venus (N = 3) 761.3631.0 15.060.3 41.761.2 54.760.7 19.760.3 36.060.3 17.061.7 118.7611.6 ... MEDMC-NT2 (N = 3) 772.0625.3 15.160.5 41.461.8 53.760.3 19.560.3 36.560.3 12.765.7 124.369.3 . .. Experiment B . . .. Day 5 . ... MEDEP-BRC5 (N = 10) 562.7622.4* 13.061.1 34.761.4* 61.661.2 23.061.1 37.461.7 150.7636.1 75.568.0 . ... MEDMC-BRC6 (N = 10) 506.9631.7 12.160.7 32.361.5 63.862.5 23.861.2 37.462.5 216.46100.5 86.1619.2 . .. Day 26 . ... MEDEP-BRC5 (N = 10) 933.5634.4 14.961.2 49.062.3 52.561.5 15.961.0 30.461.3 72.5617.3 95.166.9 . ... MEDMC-BRC6 (N = 10) 922.7626.1 14.861.0 48.761.2 52.861.2 16.061.0 30.361.4 78.3617.7 105.0610.3 . . . .. Experiment A, MEDEP-E14-Venus and MEDMC-NT2 (26107 cells/mouse) were transplanted into immuno-deficient mice (NOD-SCID) 24 hours following the induction of .. hemolysis by phenylhydrazine (60 mg/kg body weight) injection. Experiment B, MEDEP-BRC5 and MEDMC-BRC6 (26107 cells/mouse) were transplanted into C57BL/6 .. . mice 24 hours following the induction of hemolysis by phenylhydrazine (80 mg/kg body weight) injection. Day 5 and Day 26, five and twenty-six days after cell .. transplantation. RBC, red blood cell, 6104/ml. Hb, hemoglobin, g/dl. Ht, hematocrit, %. MCV, mean corpuscular volume, fl. MCH, mean corpuscular hemoglobin, pg. . .. MCHC, MCH concentration, g/dl. WBC, white blood cell, 6102/ml. Platelet, 6104/ml. .. *p,0.01 (by the Student’s t-test) .. . doi:10.1371/journal.pone.0001544.t003 RBCs derived from MEDEP are functional in vivo To confirm that the RBCs derived from the transplanted MEDEP cells are functional in vivo, we monitored the response of transplanted mice to a second induction of hemolysis. Similar to the experiments shown in Figure 4A and Table 3, a prior induction of hemolysis and subsequent cell transplantation were performed. A second induction of hemolysis was performed five days after the cell transplantation (Figure 4B). Analysis of blood count was not performed at any time point in this experiment, because collection of peripheral blood would affect the results. We observed that one out of eight mice in the group transplanted with MEDEP-E14 cells died while seven out of eight mice in the group transplanted with control cells (MEDMC-NT2) died (Figure 4B). The mice that did not receive any transplanted cells demonstrated a mortality similar to that of mice transplanted with control cells (data not shown). This result was consistent with the increased RBC number five days after cell transplantation (Figure 4A). In other words, this result indicated that RBCs derived from MEDEP cells were functional in vivo and that mice transplanted with MEDEP cells could survive the induction of severe acute anemia following a second induction of hemolysis. Concluding Remarks At present, we cannot precisely describe the exact mechanism underlying the establishment of differentiated cell lines from ES cells. Nevertheless, our results clearly indicate that we can reproducibly obtain useful erythroid cell lines from mouse ES cells. Given that differentiation strategies developed for mouse ES cells can differ from methods applied to human ES cells in many cases [33], the method we developed here may not be applied to human ES cells directly and some modifications may be necessary. However, considering the number of human ES cell lines that have been established so far, we believe that these human ES cell lines, at least in part, should exhibit the potential to produce erythroid cell lines. In addition, our results strongly suggest the possibility of establishing useful cell lines committed to specific lineages other than hematopoietic progenitors from human ES cells. The induction of terminally differentiated cells that no longer proliferate should enable clinical applications of ES cell derivatives without an associated risk of tumorigenicity. For example, RBCs lack nuclei following terminal differentiation, and thus are highly unlikely to exhibit tumorigenicity in vivo. As such, even if the original ES cells and/or their derivatives possessed abnormal karyotypes and/or genetic mutations, they may nonetheless be useful for clinical applications, provided that they can produce enucleated RBCs. In fact, although our MEDEP lines included many cells possessing abnormal karyotypes (Figure S5), the vast majority of cells in each cell line could nevertheless differentiate into mature erythroid cells (Figure 2A), including enucleated cells (Figure 2C). We showed a model of transplantation therapy using MEDEPs in this study as an application of ES cell-derivatives. On the other hand, methods to produce enucleated RBCs abundantly from human hematopoietic stem cells in vitro have recently been reported [2,3]. Therefore, once appropriate erythroid cell lines have been established, it would be possible to use such methods to produce enucleated RBCs from such cell lines in vitro. The establishment of a human erythroid cell line lacking the genes to produce the A, B, and RhD antigens would be a universal resource for clinical application, since the cell line produces O/RhD(-) RBCs which are transfusable into all individuals in theory. MATERIALS AND METHODS Mouse ES cell lines Mouse ES cell lines, E14TG2a and D3, were obtained from the American Type Culture Collection (ATCC; Monassas, VA, USA). E14TG2a and D3 were derived from 129/Ola and 129/Sv+c/+p mice, respectively. Other mouse ES cell lines were obtained from the Cell Engineering Division of RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). TT2 was derived from F1 C57BL/6 and CBA mice. BRC4, BRC5, BRC6, and BRC7 were derived PLoS ONE | www.plosone.org 8 February 2008 | Issue 2 | e1544