Figure 4.

Reversible effect of Bcr-Abl expression on erythroid differentiation. (A) ES cells were induced to differentiate by removal of LIF and cultured on OP9 layers between days 0 and 5 in the presence of Tet (100 ng/ml) to suppress Bcr-Abl expression. Differentiated cells were harvested, washed, and replated on OP9 layers in three different groups. In the first group, cells were continuously cultured in Tet between days 5 and 8, when a sample was harvested, and between days 8 and 14. In the second group, cells harvested on day 5 were washed and recultured in the absence of Tet between days 5 and 8, when the culture was sampled, and continued between days 8 and 14 without Tet. In the third group, cells from day 5 were washed and cultured between days 5 and 8 without Tet, a sample was harvested, and Tet was added back to suppress Bcr-Abl expression. A portion of each sample was extracted for Western blot analysis with anti-Abl monoclonal as described in Materials and Methods to monitor expression of Bcr-Abl at the indicated time points. (B) Cells harvested on day 14 from each of the groups described above were analyzed by FACScan for the percentage of erythroid (TER119⁺), myeloid (CD11b⁺), and other cell types. The total cell number per culture is shown. (C) Morphology of cell preparations harvested and analyzed after Giemsa staining. Magnification is ×500.