Density of the Notch ligand Delta1 determines generation of B and T cell precursors from hematopoietic stem cells

Abstract

Notch signaling regulates multiple cell fate decisions by hematopoietic precursors. To address whether different amounts of Notch ligand influence lineage choices, we cultured murine bone marrow lin(-)Sca-1(+)c-kit+ cells with increasing densities of immobilized Delta1(ext-IgG) consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG1. We found that relatively lower densities of Delta1(ext-IgG) enhanced the generation of Sca-1(+)c-kit+ cells, Thy1(+)CD25+ early T cell precursors, and B220(+)CD43(-/lo) cells that, when cocultured with OP9 stroma cells, differentiated into CD19+ early B cell precursors. Higher densities of Delta1(ext-IgG) also enhanced the generation of Sca-1(+)c-kit+ precursor cells and promoted the development of Thy1(+)CD25+ cells, but inhibited the development of B220(+)CD43(-/lo) cells. Analyses of further isolated precursor populations suggested that the enhanced generation of T and B cell precursors resulted from the effects on multipotent rather than lymphoid-committed precursors. The results demonstrate the density-dependent effects of Delta1 on fate decisions of hematopoietic precursors at multiple maturational stages and substantiate the previously unrecognized ability of Delta1 to enhance the development of both early B and T precursor cells.

Figure 1.

Proliferation and differentiation of LSK cells during culture with various Delta1^ext-IgG densities. (A) Total number of cells (▾) generated from LSK cells after 14 d of incubation with Delta1^ext-IgG plated at increasing concentrations. (B) Number of cells exhibiting an immature (Sca-1⁺c-Kit⁺; ⋄) or myeloid (Gr1⁺ and/or F4/80⁺; ▴) phenotype. (C) Cells with an early T cell (Thy1⁺CD25⁺; ○) or early lymphoid (B220⁺ CD43^−/lo; ▪) phenotypes were determined based on cell count and FACS analysis. (D) The data shown represent the mean ± SEM of three experiments. The numbers in the corners of the flow cytometry plots represent the percentage of gated events within that quadrant.

Figure 2.

Induction of pre-T α and CD3 ɛ expression is dependent on the density of Delta1^ext-IgG. (A) CD3ɛ and (B) pre-Tα gene expressions by LSK cells after culture for 28 d with different densities of Delta1^ext-IgG determined by quantitative RT-PCR. Data shown represent the ratio of gene expression compared with the housekeeping gene Rpl7.

Figure 3.

T and B cell potential of Delta1^ext-IgG–cultured B220⁺CD43^−/lo or B220⁻ populations. After the culture of LSK cells for 21 d with different densities of Delta1^ext-IgG, B220⁺CD43^−/lo (A) or B220⁻ (B) cells were isolated by FACS and transferred to cultures containing OP9 stromal cells or 10 μg/ml of Delta1^ext-IgG. (C) Early B (B220⁺CD19⁺) or early T (Thy1⁺CD25⁺) cell phenotypes were determined with FACS analysis. The numbers in the corners of the flow cytometry plots represent the percentage of gated events within that quadrant.

Figure 4.

Differentiation of LSK-SP, LSK-Flt3⁺, and CLP cells during culture with various Delta1^ext-IgG densities. LSK-SP, LSK-Flt3⁺, and CLP cells isolated by FACS from marrow were incubated with Delta1^ext-IgG plated at increasing concentrations. LSK-SP (A) and LSK-Flt3⁺ (B) cells were incubated for 14 d and CLP (C) cells were incubated for 3 d. Numbers of generated myeloid (Gr1⁺ and/or F4/80⁺; ▴), early T cell (Thy1⁺CD25⁺; ○), or early lymphoid (B220⁺ CD43^−/lo; ▪) cells were determined based on cell count and FACS analysis.