Single-donor predominant engraftment after transplantation of 2 UCBs as a mixture of total mononuclear cells. (A) Donor origin of cord blood cells engrafted in NOD/SCID mice as determined by PCR-SSOP. Combinations of 2 UCB units were made with varying degrees of HLA disparity, and total MNCs corresponding to 5 × 104 CD34+ cells from each unit were infused into irradiated NOD/SCID mice in single (CH1-CH4) or double (CH1 + 2, CH3 + 4) transplantations. Genomic DNA harvested from recipients' bone marrow was subjected to PCR amplification by DPB1 locus-specific primers and were hybridized to allele-specific probes (P1-P4), where positive controls were DNA from donor cells (cont1-cont4). Shown are the results from 2 cohorts of double transplantations with either a full match at 6 loci (HLA-A, B, DR) (left) or 2 mismatches (HLA-B, DR) (right). (B) Profiles of RQ-PCR for the human STR to determine donor ratio of engrafted cells. Genomic DNA was harvested from mouse bone marrow and subjected to RQ-PCR analysis on 16 human STR markers. Shown are the results from a representative marker in each cohort. CB-A and CB-B were artificially nominated for the dominant and nondominant cord unit, respectively. (C) Absence of additive engraftment in transplantation of 2 UCB units as a mixture of total mononuclear cells. Overall engraftment levels of transplanted cord blood cells in NOD/SCID mice were measured by staining harvested mouse bone marrow with human cell–specific anti-CD45/CD71, as described in “Materials and methods.” Shown are the mean engraftment levels ± SEM from single or double cord transplantation (n = 22) from 9 cohorts.
