Effect of inducible WT and R459M SHP-1 expression on the growth of ES cells differentiating in coculture with OP-9 stromal cells. (A) WT and R459M SHP-1 ES cell transfectants were placed in coculture for 4 days in the presence (+ TET) or absence (NO TET) of 500 ng/mL Tet. OP-9 cells were removed by differential adhesion, and the numbers of differentiated ES cell derivatives obtained were determined. Data from experiments using WT (n = 4; ▦) and R459M (n = 4; □) SHP-1–expressing transfectants are shown. (B) Cells derived as in panel A in the presence of Tet were placed in coculture for another 6 days in the presence or absence of 500 ng/mL Tet, and the numbers of cells obtained were determined. Data from experiments using WT (n = 3) and R459M (n = 6) SHP-1–expressing transfectants are shown. For each experiment, the fold change in the number of cells obtained -Tet compared with +Tet was calculated, the average was taken, and standard errors are shown. Data were subjected to paired Student t test (^***P < .005; ^**P < .05). (C) Dark-field digital photomicrograph of HCAs derived from hemopoietic progenitors generated from R459M SHP-1/OP-9 coculture in the presence or absence of 500 ng/mL Tet for the duration of the coculture and HCA. Arrows indicate the positions of granulocyte/macrophage colonies, original magnification ×15. (D, left) Cells in HCAs generated from R459M SHP-1 transfectants differentiated in EB culture were removed from the HCA, and cytospins were prepared. Cells were stained with May-Grünwald/Giemsa stain, and the relative proportions of myeloid cell types and erythrocytes were determined (right). Original magnification ×400, a 40×/0.65 objective lens was used. Results are the averages of triplicate counts, and error bars are standard deviations. Data were subjected to paired Student t test (^**P < .05).