Detection of human repopulating cells in NOD/SCID mice transplanted with CD34⁺CD38⁻Lin⁻cells cultured in the presence of hDelta-1.

(A) Representative Southern blot analysis of the genomic DNA extracted from the BM of individual mice transplanted with 4 aliquots of 625 cells or 2500 CD34⁺CD38⁻Lin⁻cells seeded into individual wells and cultured for 4 days in the absence or presence of hDelta-1. Genomic DNA extracted from BM of mice was digested with EcoR1, separated on agarose gels, and probed with human chromosome 17–specific α-satellite probe as shown previously.54 Human:mouse DNA mixture controls shown were used to measure the level of engraftment. (B) Multilineage differentiation of human repopulating cells engrafting the BM of NOD/SCID mice following hDelta-1 treatment and control-treated repopulating cells. Cells from the BM of positively engrafted mice were stained with various human-specific monoclonal antibodies and analyzed by flow cytometry. (i) Forward- and side-scatter properties were used to gate live cells in R1 for analysis. (ii) Isotype control for nonspecific IgG staining for PE and FITC fluorescence. Cells expressing the human pan-leukocyte marker CD45 were gated (not shown) and analyzed for expression of multiple-lineage human hematopoietic markers. (iii) Pan–B-cell markers CD19 and CD20 for cells of the lymphoid lineage. (iv) Myeloid markers CD15 and CD33 for immature subsets. (v) Cell surface markers CD34 and CD38 for immature subsets.