ESC-Derived Erythroid Cells Figure 4. Amelioration of anemia by transplantation of MEDEP. (A) MEDEP-E14 cells (26107 cells/mouse) were transplanted into an immuno- deficient mouse (NOD-SCID) 24 hours after the induction of hemolysis by phenylhydrazine (60 mg/kg body weight) injection. Day 5 and Day 26, five and twenty-six days after cell transplantation. RBC, red blood cell. White bars (n = 10) and black bars (n = 14), the data obtained from the mice transplanted with control cells and MEDEP-E14 cells, respectively. Values are mean6S.D. * p,0.01 (by the Student’s t-test) (B) Increased survival of mice transplanted with MEDEP cells following induction of severe acute anemia. MEDEP-E14 cells (26107 cells/mouse) were transplanted into an NOD-SCID mouse 24 hours following the first induction of hemolysis by phenylhydrazine (60 mg/kg body weight) injection. Five days following the cell transplantation, the second induction of hemolysis by phenylhydrazine (80 mg/kg body weight) injection was performed. Statistical analysis was performed using the chi-square test. (A, B) Control cell, mast cell line derived from mouse ES cells (MEDMC-NT2) (Figures S1 and S2). MEDMC-NT2 cells (26107 cells/mouse) were transplanted similarly as a control experiment. doi:10.1371/journal.pone.0001544.g004 The transplantation of MEDEP-E14-Venus and MEDEP- BRC5 cells also ameliorated anemia compared to the control (Table 3). However, the transplantation of MEDEP-BRC5 cells appeared to be less effective for amelioration of anemia than MEDEP-E14 cells (compare Figure 4A and Table 3). Given that the in vitro proliferation activity of MEDEP-BRC5 cells was lower than that of MEDEP-E14 cells (Figure 1B), the in vivo proliferation activity of MEDEP-BRC5 cells might have also been lower than that of MEDEP-E14 cells. In addition, hemoglobin synthesis in MEDEP-BRC5 might have been less efficient than that in MEDEP-E14 (Figure 2B and Table 3). Immunogenicity of human ES cell derivatives is one of the potential obstacles to the clinical use of them [28,29]. In fact, transplanted MEDEP cells could not ameliorate acute anemia in mouse strains other than those from which each individual line was derived or in immuno-deficient mice, suggesting immunolog- ical rejection by the heterologous strains. Hence, if human erythroid cell lines could be established, the clinical application of such cell lines may require many cell lines expressing the different major histo-compatibility (MHC) antigens. Lack of tumorigenicity of MEDEP Approximately three months (82 days) after transplantation, Venus- positive cells were absent from the bone marrow and spleen of mice transplanted with MEDEP-E14-Venus cells (Figure S8). In addition, although we observed all other transplanted mice up to 6 months after transplantation, no tumor was observed in MEDEP-trans- planted mice or MEDMC-transplanted control mice (data not shown). Furthermore, subcutaneous transplantation of MEDEP cells (26107 cells/injection site) did not give rise to any tumors, whereas subcutaneous transplantation of the same number of parent ES cells led to the formation of a teratoma (data not shown). Nevertheless, if human erythroid cell lines were established, the tumorigenic potential of those cell lines should be thoroughly analyzed prior to use in the clinic [30,31]. It may be advisable to engineer such cells so that they could be eliminated should a malignant phenotype arise for any reason [32]. PLoS ONE | www.plosone.org 7 February 2008 | Issue 2 | e1544