Effects of AML1dC on c-Mpl expression on hematopoietic progenitor cells
and megakaryocytes and megakaryopoiesis. A, experimental design
using the OP9 system. ES cells were deprived of leukemia inhibitory factor and
cultured on OP9 cells for 4.5 days. Then Flk-1+ cell were sorted,
replated onto OP9 cells, and cultured with mSCF and hTPO (for the analysis of
c-Mpl expression in hematopoietic stem/progenitor cells) or only hTPO (for the
analysis of megakaryocytic differentiation) with or without Tet for the time
indicated. B, c-Mpl expression of nonadherent cells was examined by
the direct immunofluorescence method on day 8.5 and day 12.5. The percentage
of each fraction is indicated. The relative frequency of GFP-
fraction in cultured cells with Tet and the relative frequency of
GFP+ fraction in cultured cells without Tet were shown in
parentheses. C, c-Mpl expression on early hematopoietic
cells that derived from WT and AML1+/- ES cells on day 8.5.
D and E, after 12.5-day cultures with TPO, megakaryocytic
cells, which derived from ES cells expressing AMLdC, were subjected to
morphological analysis (D), and DNA content analysis by propidium
iodide staining (E).