Notch signaling regulates multiple cell fate decisions by hematopoietic precursors. Previously, we found that endogenous Notch signaling in cultures of murine hematopoietic precursors (Lin-Sca-1+ c-Kit+) leads to a multi-log increase in the number of Sca-1+ c-Kit+ cells, inhibition of myeloid differentiation, and promotion of T/NK differentiation. To activate Notch signaling in those studies, a single dose (10μg/ml) of engineered Notch ligand consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG1 (Delta1ext-IgG) was immobilized to the plastic tissue culture surface. To investigate quantitative effects of Notch signaling, bone marrow Lin-Sca-1+ c-Kit+ (LSK) cells were cultured with plates coated with increasing concentrations of Delta1ext-IgG in media supplemented with 20% FBS, SCF (100 ng/mL), Flt3L (100 ng/mL), IL6 (100ng/mL) and IL11 (10ng/mL). LSK cells cultured for 14 days with control human IgG1 underwent terminal myeloid differentiation (determined by expression of GR1 and F4/80) with no further increase in cell number, whereas at all densities of Delta1ext-IgG there was approximately a 3 log greater number of cells than in control cultures. Furthermore, the portion of cells that maintained Sca-1 and c-Kit expression increased at greater densities of Delta1ext-IgG (10%, 32%, 77%, 71%, 71% and 71% for plates coated with ligand at 0.6, 1.25, 2.5, 5, 10 and 20 μg/ml, respectively, and 5% for human IgG1 control at 10μg/ml), whereas the portion of cells undergoing myeloid differentiation decreased at greater ligand densities (48%, 33%, 5%, 3%, 3% and 3% respectively, and 40% for control). In contrast, a substantial increase in the portion of cells expressing B220+ was observed at relatively low densities of Delta1ext-IgG (30% at 0.6 μg/ml and 19% at 1.25 μg/ml) compared to control (4%), but was no longer evident with further increases in ligand density (1.8%, 2%, 1.2%, m1.6% at 2.5, 5, 10 and 20 μg/ml respectively). Furthermore, promotion of early T cell differentiation was observed in ligand containing cultures with the generation of increased number of cells co-expressing Thy1.2 and CD25 (14%, 24%, 22% and 24% at 2.5, 5, 10 and 20 μg/ml respectively). Further evidence for T cell commitment was established by quantitative RT-PCR in which increased expression of CD3ε and pre-Tα was observed by 28 days of culture. Thus these studies demonstrate that culture with different densities of the Notch ligand, Delta1ext-IgG results in differential cell fate outcome with inhibition of myeloid differentiation and promotion of early T cell induction that is maximal at high ligand densities and of B220+ cells at relatively lower densities.
Expression and regulation of C/EBPα in normal myelopoiesis and in malignant transformation
