Density-Dependent Regulation of Hematopoietic Stem Cell Fate by the Notch Ligand, Delta1.

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 (Delta1^ext-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 Delta1^ext-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 Delta1^ext-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 Delta1^ext-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 Delta1^ext-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, Delta1^ext-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.