Culture of Lin⁻Sca-1⁺c-kit⁺hematopoietic cells with immobilized Notch ligand, Delta1^ext-IgG, and hematopoietic growth factors.

(A) Schematic diagram of engineered Notch ligand consisting of the extracellular domain of Delta1 fused to the Fc portion of human IgG1 (Delta1^ext-IgG). Delta1^ext-IgG includes the Delta-Serrate-Lag1 (DSL) domain and epidermal growth factor (EGF) repeats. The control molecule consists of a signal peptide fused to the Fc portion of human IgG1 (Control^IgG). (B) Total number of cells generated over time was determined during culture with 4GF and I-Delta1^ext-IgG (♦); 4GF, IL-7, and I-Delta1^ext-IgG (●); 4GF and I-Control^IgG (▪); 4GF and NI-Delta1^ext-IgG(▴). (C-E) Morphology and phenotype of cultured cells. Wright-Giemsa–stained cytospin preparations (far left panels and fluorescence histograms [right panels]) of cells cultured for 14 days with 4GF and I-Delta1^ext-IgG (C), I-Control^IgG(D), or NI-Delta1^ext-IgG (E). Cells were stained with monoclonal antibodies that recognize Sca-1, c-Kit, GR-1, or CD25 epitopes. Original magnification, × 400. X-axis, log fluorescence intensity; y-axis, cell number; solid line, staining with test antibody; dotted line, staining with isotype-matched control antibody.