ESC-Derived Erythroid Cells .. . Table 1. Culture protocol. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... Phase Culture period Feeder cells Attached cells Detached cells Specific factors used . ... I Day 0 OP9 cells (Start) (Start) VEGF, IGF-II . ... II Day 4 Change to new cells Discarded a Re-cultured b SCF, EPO, IL-3, Dex .... II Day 7 No change Remained c Re-cultured d SCF, EPO, IL-3, Dex ... II Day 10,e Change or No change f Remained or Discarded g Re-cultured h SCF, EPO, IL-3, Dex . ... III Day 60,i (-) j (-) k Re-cultured h SCF, EPO, IL-3, Dex .... IV Day 120,l (-) j (-) k Re-cultured h Essential factors l . . .. To induce hematopoiesis, 56105 ES cells were cultured on feeder cells with cytokines in two 100 mm-dishes, with 2.56105 ES cells per dish. Phase I,IV, four different .. . phases of culture. Attached cells and Detached cells, the cells derived from ES cells and attached to feeder cells or detached from feeder cells. VEGF, vascular endothelial .. growth factor. IGF-II, insulin-like growth factor-II. SCF, stem cell factor. EPO, erythropoietin. IL-3, interleukin-3. Dex, dexamethasone. .. . a, the attached cells were discarded together with the used feeder cells. b, the detached cells collected from two dishes were cultured again on new OP9 cells in a ... 100 mm-dish. c, the attached cells were cultured further without any treatment. d, all detached cells collected from a dish were cultured again. e, medium changes were .. performed twice a week. f, when the attached cells reached approximately 80% confluence, feeder cells were changed to new OP9 cells. g, when the feeder cells were .. changed to new cells, the attached cells were discarded together with the used feeder cells. h, all detached cells collected from a dish were cultured again, or a portion . .. of detached cells were cultured again and other detached cells were subjected to analyses or discarded. i, approximately as of Day 60 we started to try the culture in the .. absence of feeder cells using a portion of the detached cells, simultaneously continuing the culture in the presence of feeder cells as the Phase II culture. j, no feeder . .. cells were used in the Phase III and IV culture. k, the cells attached to the dish were barely detected. l, approximately as of Day 120 the essential factor(s) for proliferation . . was evaluated, and then each cell line was cultured in the presence of the essential factor(s) alone. . .. doi:10.1371/journal.pone.0001544.t001 cases, the induced cells failed to proliferate within two months of the initial induction of differentiation from ES cells (Table 2). Induced cells that could proliferate continuously for approximately two months (60 days) were subsequently cultured in the absence of OP9 cells and in the presence of hematopoietic humoral factors (Table 1). Cells that could proliferate in the absence of OP9 cells were cultured further. All established cell lines (Table 2) acquired independency from OP9 cells within three months of the initial induction of differentiation from ES cells. Approximately four months after the initial induction of differentiation of the cells, we evaluated the factors that were essential for the proliferation of each cell line (Table 1). After this evaluation, each cell line was cultured in the presence of these essential factors with medium changes every two or three days. In addition to the method described in Table 1 (Method A), we developed Method B in which the use of IL-3 was excluded from Method A through all procedures (Table 2). We attempted long term culture of 63 lines, 51 lines with Method A and 12 lines with Method B, and succeeded in establishing five independent immortalized cell lines, 4 lines with Method A and 1 line with Method B (Table 2). These five cell lines could proliferate continuously for more than one year. Morphological and flow cytometric analyses suggested that three of these lines were erythroid in nature, as shown below, and that the other two lines were mast cell-like (Figures S1 and S2). We designated the erythroid cell lines MEDEP (mouse ES cell-derived erythroid progenitor line) and the mast cell-like cell lines MEDMC (mouse ES cell-derived mast cell line). MEDEP-E14, MEDEP- BRC4, and MEDEP-BRC5 were derived from E14TG2a, BRC4, and BRC5 mouse ES cell lines, respectively. The presence of IL-3 in the culture medium (Method A) may not be necessary for the establishment of erythroid cell lines, as we were able to establish one erythroid line, MEDEP-BRC4, following culture of the cells in the absence of IL-3 (Method B) (Table 2). MEDEP cells could proliferate from single cells following sorting by flow cytometry (data not shown). MEDEP-E14 and MEDEP-BRC5 cells retained morphological characteristics of erythroid cells (Figure 1A) and cytokine depen- dency (Figure 1B) after cloning. MEDEP-E14 and MEDEP-BRC5 were dependent on erythropoietin (EPO) and stem cell factor (SCF), respectively (Figure 1B). Although MEDEP-BRC5 appeared to respond to EPO (Figure 1B), it could not proliferate long term in the presence of EPO alone (data not shown). MEDEP-BRC4 also showed morphological characteristics of erythroid cells (Figure S3) and could proliferate most efficiently in the presence of SCF, EPO, and dexamethasone (Figure S3). The cytokine dependency of these cell lines has not changed for more than one year after the induction of their differentiation from ES cells. RT-PCR analyses demonstrated that all MEDEP lines expressed genes specific for erythroid cells: GATA-1, EKLF (Erythroid Kru¨ppel-like factor) and EPOR (erythropoietin receptor) (Figure 1C and Figure S4). In addition, all MEDEP lines expressed a- and b-globin, but not c-, e-, or f-globin (Figure 1C and Figure S4), indicating that they were adult and not primitive erythroid progenitor cells. Since the induction of definitive erythropoiesis, i.e., the induction of adult type erythroid cells, from mouse ES cells . .. Table 2. The number of trials to establish cell lines and the . . number of established cell lines. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Method A . . ..... cNealmlleineof ES oNfutmribaler lNinuember of established cell Designation . ... E14TG2a 10 1 MEDEP-E14 . ... D3 3 0 .... TT2 3 0 ... BRC4 7 0 . ... BRC5 10 1 MEDEP-BRC5 . ... BRC6 6 1 MEDMC-BRC6 ... BRC7 6 0 . ... NTES2 6 1 MEDMC-NT2 . .. Method B . . ..... cNealmlleineof ES oNfutmribaler cNeullmlibneer of established Designation . ... BRC4 4 1 MEDEP-BRC4 . ... BRC5 2 0 .... BRC6 4 0 ... BRC7 2 0 . . .. . Method A, the method described in Table 1. Method B, the use of IL-3 was .. excluded from Method A through all procedures. .. . doi:10.1371/journal.pone.0001544.t002 PLoS ONE | www.plosone.org 2 February 2008 | Issue 2 | e1544