from C57BL/6 mice. NTES-2 was established following transfer of nuclei of cells derived from 129 mice into oocytes. ES cells were maintained in an undifferentiated state on mouse embryonic fibroblasts (MEFs) in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM; SIGMA, St Louis, MO, USA) and Ham’s nutrient mixture F-12 (SIGMA) supplemented with 1,000 U/ml leukemia inhibitory factor (LIF; CHEMICON International Inc., Temecula, CA, USA), 0.1 mM non-essential amino acids, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, and 20% KnockOutTM Serum Replacement (KSR; Invitrogen, Carlsbad, CA, USA). Before MEFs were used as feeder cells, they were irradiated with c-rays (50 Gy). Establishment of hematopoietic cell lines from mouse ES cell lines The feeder cell line, OP9 [23], used to induce hematopoietic differentiation of ES cells [5,6] was obtained from the Cell Engineering Division of RIKEN BioResource Center and was cultured in Minimum Essential Medium-a (MEM-a; SIGMA) containing 20% fetal bovine serum (FBS; SIGMA). Before OP9 cells were used as feeder cells, they were irradiated with c-rays (50 Gy). The basal medium used throughout long term culture was Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen) containing the following materials: 15% FBS (SIGMA); 10 mg/ ml bovine insulin, 5.5 mg/ml human transferrin, and 5 ng/ml sodium selenite (ITS liquid MEDIA supplement; SIGMA); 50 mg/ ml ascorbic acid (SIGMA); 0.45 mM a-monothioglycerol (SIG- MA); 100 unit/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine (PSQ; Invitrogen). Specific factors used were as follows: mouse vascular endothelial growth factor (VEGF, 20 ng/ml; R&D systems, Minneapolis, MN, USA), mouse insulin-like growth factor-II (IGF-II, 200 ng/ ml; R&D systems), mouse stem cell factor (SCF, 50 ng/ml; R&D systems), human erythropoietin (EPO, 5 unit/ml; KIRIN Brewery Company, Tokyo, Japan), mouse interleukin-3 (IL-3, 10 ng/ml; R&D systems), and Dexamethasone (Dex, 1026 M; SIGMA). We developed two different methods to establish hematopoietic cell lines from mouse ES cells, Method A and Method B. Method A is described in Table 1. In Method B, the use of IL-3 was excluded from Method A through all procedures. Viable cell number was assessed using an automated cell counter and an assay based on the trypan blue dye exclusion method, ViCellTM (BECKMAN COULTER, Fullerton, CA, USA). Morphology of the established cell lines was analyzed after Wright-Giemsa (Sysmex International, Kobe, Japan) staining. Reverse transcription-polymerase chain reaction (RT-PCR) A semi-quantitative RT-PCR was performed as described previously [24]. PCR was carried out with recombinant Taq polymerase (TaKaRa Bio Inc., Otsu, Shiga, Japan). Cycling parameters were as follows: denaturation at 94uC for 30 sec, annealing at 55uC for 30 sec, and extension at 72uC for 30 sec. PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide staining. Amplification of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control in the PCR. The sequences of the PCR primers were as follows: for mouse Oct-3/4, the sense primer was 59-ACC CAG GCC GAC GTG GGG CT-39 and the antisense primer was 59-TTC TGG CGC CGG TTA CAG AAC CA-39 (365-bp PCR product); for mouse Nanog, the sense primer was 59-TAC CTC AGC CTC CAG CAG AT-39 and the antisense primer was 59-CCT CCA AAT ESC-Derived Erythroid Cells CAC TGG CAG-39 (460-bp PCR product); for mouse GATA-1, the sense primer was 59-ACA GGT CAC TAC CTG TGC AAT GCC-39 and the antisense primer was 59-CCT GAC AGT ACC ACA GGT CCT AG-39 (463-bp PCR product); for mouse EKLF (Erythroid Kru¨ ppel-like factor), the sense primer was 59-TAT GGG CTG CTG TCG GGA TAC CC-39 and the antisense primer was 59-TCA GAG GTG ACG CTT CAT GTG CAG -39 (507-bp PCR product); for mouse EPOR (erythropoietin receptor), the sense primer was 59-ATC CAT ATC AAT GAA GTA GTG CTC-39 and the antisense primer was 59-CCA CAG CTG GAA GTT ACC CTT GTG-39 (513-bp PCR product); for mouse a- globin, the sense primer was 59-CTC TCT GGG GAA GAC AAA AGC-39 and the antisense primer was 59-GGT GGC TAG CCA AGG TCA CCA-39 (334-bp PCR product); for mouse b (b- major)-globin, the sense primer was 59-GAT GCT GAG AAG TCT GCT GTC-39 and the antisense primer was 59-CTG GAA GGC AGC CTG TGC AGC-39 (381-bp PCR product); for mouse c (b-H1)-globin, the sense primer was 59-CTC AAG GAG ACC TTT GCT CA-39 and the antisense primer was 59-AGT CCC CAT GGA CTC AAA GA-39 (265-bp PCR product); for mouse e-globin, the sense primer was 59-GGA GAG TCC ATT AAG AAT CTA-39 and the antisense primer was 59-CTG TGA ATT CAT TGC CGA AGT-39 (157-bp PCR product); for mouse f-globin, the sense primer was 59-GCT CAG GCC GAG CCC ATT GG-39 and the antisense primer was 59-TAG CGG TAC TTC TCA GTC AG-39 (371-bp PCR product); and for mouse GAPDH (glyceraldehyde-3-phosphate dehydrogenase), the sense primer was 59-GTC TTC ACC ACC ATG GAG AAG-39 and the antisense primer was 59-GCC ATC CAC AGT CTT CTG GGT- 39 (270-bp PCR product). Flow cytometry Cells were stained with monoclonal antibodies (MoAbs) and analyzed by FACS Calibur (BD Biosciences, San Jose, CA, USA) or sorted by FACSVantage SE (BD Biosciences). The following MoAbs were purchased from BD Biosciences: a fluorescein isothiocyanate (FITC)-conjugated MoAb against mouse CD71 (transferrin receptor), a phycoerythrin (PE)-conjugated MoAb against TER119 (a cell surface antigen specific for mature erythroid cells), an allophycocyanin (APC)-conjugated MoAb against mouse c-Kit (receptor for stem cell factor), a PE- conjugated MoAb against mouse CD71, and a biotin-conjugated MoAb against TER119. APC-conjugated streptavidin (BD Biosciences) was used to detect biotin-conjugated MoAb. To distinguish nucleated and enucleated cells, we used the SYTO85 nuclear stain (Invitrogen). Cell viability was monitored by propidium iodide (SIGMA) staining. Flow cytometry data were analyzed using FlowJo (Tree Star Inc., Ashland, OR, USA) analysis software. Morphology of the sorted cells was analyzed following Wright-Giemsa (Sysmex International) staining. Mice Eight-week-old female NOD/shi-scid Jic and C57BL/6NCrj mice were purchased from CLEA Japan (Tokyo, Japan) and Charles River Laboratories Japan (Yokohama, Kanagawa, Japan), respec- tively. Mice were used within a week of delivery in all experiments. All experimental manipulations of mice were approved by the Institutional Animal Care and Use Committee of the RIKEN Tsukuba Institute. Induction of acute anemia in mice Acute anemia was induced by hemolysis following intraperitoneal injection of phenylhydrazine (Wako, Osaka, Japan), a chemical PLoS ONE | www.plosone.org 9 February 2008 | Issue 2 | e1544