Representative FACS profile of primary, secondary, and tertiary engraftment in the BM of NOD/SCID mice after injection of human CB CD34⁺ cells at start of cultures and of their progeny at different times of fractionated long-term expansion. (A) Isotype control. (B) Representative FACS profile of marrow cells from a NOD/SCID mouse that had not received a transplant. (C) Primary, secondary, and tertiary engraftment was evaluated in the BM of a NOD/SCID mouse that was injected 8 weeks previously with 3 × 10⁵ CB CD34⁺ cells or 20 × 10⁶ unseparated BM cells of primary or a secondary mouse. The level of human engraftment in the mouse BM was evaluated by FACS analysis (a positive mouse was defined by the presence of ≥ 0.1% human CD45⁺, CD71⁺, and GpA⁺ cells on total BM cells), confirmed by DNA analysis as described in “Patients, materials, and methods.” Total engraftment (CD45⁺ cells and GpA⁺ cells) was performed within the total, unseparated BM cells in individual NOD/SCID mice.