Competitive Repopulation Assay for Human Stem Cell Engraftment in NOD- scid IL-2rγ nul Mice.

Umbilical cord blood (UCB) is a source of stem cells for hematopoietic transplantation (Laughlin, et al. NEJM2004; 351:2265) but application in adults is limited by marginal stem cell numbers. Combining cord blood units has shown promise in achieving engraftment (Barker, et al. Blood2002; 100:41a [abs #142]) without crossed immunologic rejection. Short-term hematopoiesis showed contributions from both cords, but long-term engraftment derived predominantly from just one cord. To study this process we have developed an in vivo pre-clinical competitive repopulation assay using NOD- scid IL-2rγ null mice that support high levels of human UCB engraftment (Shultz, et al, J. Immunol.2005, 174:6477). NOD- scid IL-2rγ null mice were transplanted with T-cell depleted UCB containing 3x10 4 HLA-A2 + CD34 + cells, 3x10 4 HLA-A2 − CD34 + cells, or a 1:1 mixture (6x10 4 CD34 + cells). Human hematopoietic chimerism, measured by human CD45 and HLA-A2 expression, was quantified in bone marrow 6 and 12 weeks after transplantation. Variable mixed engraftment within individual as well as between 6 separate experiments was observed. At both 6 and 12 weeks, mixed contribution from both cords was achieved although rarely in a 1:1 ratio despite the infusion of equivalent CD34 + cell numbers. HLA-A2 expression did not provide a selective advantage. The complete dominance of one cord observed in the clinical report and in animal studies (Yahata, et al. Molecular Therapy2004; 10: 882) suggests an important role for the immune activity of UCB T-cells in this competitive process, a hypothesis we are currently testing. This model has a rapid turnaround and can test multiple permutations, providing a preclinical vehicle for characterizing optimal combinations of cord cells and for testing conditions that allow one cord to ultimately prevail.