Development of G1ME cells in Gata1^- chimeric embryos. (A) Experimental approach. Chimeric embryos were prepared by injecting Gata1^- or wt ES cells into wt host blastocysts. Fetal liver hematopoietic cells from day 13.5 chimeric embryos were analyzed by FACS or cultured on OP9 cells with Tpo according to the same conditions used to derive G1ME cells from ES cells (Figure 1). The polymorphic cell-surface marker Ly9.1 was used to facilitate tracking of ES-cell donor-derived hematopoietic cells. Donor ES cells (strain 129) express Ly9.1 and CD45.2 (not shown), whereas host (C57/BL6) blastocyst-derived cells express Ly9.2 and CD45.1 (not shown). (B) Flow cytometry analysis of E13.5 fetal livers. Gata1^- chimeric embryos contain an expanded population of donor-derived (Ly9.1) cells that are lineage negative, CD41⁺, and cKit⁺, identical to the surface phenotype of G1ME cells derived from in vitro differentiation of Gata1^- ES cells, as described in Figure 1. Representative studies of fetal livers from wt and Gata1^- chimera are shown. The wt chimeric fetal liver was 30% Ly9.1⁺, and the Gata1^- chimeric fetal liver was 23% Ly9.1⁺. (C) CD41⁺ cKit⁺ cells as a percentage of lin^- fetal liver cells. As indicated on the x-axis, embryos prepared from Gata1^- or wt ES-cell-injected blastocysts were termed chimeric only if donor-derived Ly9.1 hematopoietic cells were detected in fetal livers. Levels of chimerism ranged from 2% to 23% in Gata1^- chimeric animals, and 30% to 47% in wt chimeric fetal livers. (D) Additional cell-surface marker expression comparing G1ME cells (top) and E13.5 chimeric fetal livers (middle and bottom). Fetal livers from Gata1^- or wt chimeric embryos were analyzed 4 days after expansion on OP9 stroma. All cells shown are lin^-, IL7Rα^-, and Sca1^-. Left panels show the detection of donor cells (Ly9.1⁺). Middle panels show cKit and CD41 expression in Ly9.1⁺ cells; these are analyzed further for FcγR and CD9 expression in the right panels. Percentages in the right panels refer to lin^-, IL7R^-, Sca1^-, CD9⁺, FcγR^lo, CD41⁺, and ckit⁺ cells within the Ly9.1⁺ population.