Intra–bone marrow transplantation facilitates pauci-clonal human hematopoietic repopulation of NOD/SCID/β2m−/− mice

Objective Intra–bone marrow transplantation (IBMT) has been shown to improve the limit of detection of primitive human SCID-repopulating cells (SRC) in NOD/SCID mice when compared to intravenous transplantation. We sought to further refine detection of SRC by comparing NOD/SCID mice to the more sensitive NOD/SCID/β2m−/−strain as IBMT recipients of limiting numbers of purified primitive human hematopoietic cells. Materials and Methods Purified human Lin−CD34+CD38− cells at limiting doses were delivered by IBMT into NOD/SCID and NOD/SCID/β2m−/− strains of recipient mice. Six weeks posttransplantation, injected and noninjected bones were analyzed separately for multilineage human hematopoietic chimerism. Results NOD/SCID/β2m−/− mice are superior recipients for IBMT and show a trend toward increased levels of human hematopoietic engraftment. In addition, in contrast to NOD/SCID recipients, NOD/SCID/β2m−/− mice were reconstituted with as few as five highly purified cells, indicative of pauci-clonal repopulation. Analysis of injected and noninjected bones demonstrated that engrafting cells were capable of in vivo migration and expansion. Although SRC hematopoietic reconstitution of NOD/SCID mice is commonly lymphoid-dominant, multilineage analysis of separate bone sites following IBMT of purified cells revealed that a subset of mice was repopulated with a myeloid-dominant graft in at least one bone site, revealing that SRC are developmentally heterogeneous among Lin−CD34+CD38− cells and capable of distinct differentiation potential. Conclusion IBMT into NOD/SCID/β2m−/− mice provides a highly sensitive experimental transplantation assay for the detection of human hematopoietic repopulating cells and demonstrates that Lin−CD34+CD38− cells are more highly enriched for human SRC than originally predicted.