Cell separation improves the sensitivity of detecting rare human normal and leukemic hematopoietic cells in vivo in NOD/SCID mice

Background This report describes a novel cell-separation procedure developed to improv e detection and analysis of rare human hematopoietic populations, obtained from NOD/SCID mice engrafted with normal and/or leukemic stem cells. Methods In preliminary experiments, artificial mixtures of murine and human BM cell s were labeled with a combination of Abs specific for murine hematopoietic cells, prior to immunomagnetic negative selection using StemSep. In subsequ ent experiments, BM was harvested from individual NOD/SCID mice transplante d 6-12 weeks earlier with either human cord blood or primary CML cells and a similar immunomagnetic selection procedure was applied to enrich human ce lls present. Results Application of this selection procedure to mixtures of murine and human hem atopoietic cells using anti-mouse CD45 and Ter-119 allowed a > 1000-fold de pletion of murine cells with > 50% recovery of human cells, including proge nitors. This level of depletion and recovery were found to be reproducible for NOD/SCID mice transplanted and engrafted with human cord blood stem cel ls, thus facilitating detection of human progenitors, including colony-form ing cells (CFC) and LTCIC. For NOD/SCID mice previously transplanted with C ML cells, this procedure increased the sensitivity of detective rare human cell subsets by up to > 100-fold. This, in turn, improved the sensitivity o f RT-PCR for BCR-ABL and made possible the identification by FACS of variou s minor subsets of human cells, including CD34(-)CD19/20(+) B-lineage cells , CD34(+) progenitors, mature CD15(+) myeloid cells and CD3(+) T cells pres ent in the mice. Discussion This simple cell-depletion procedure should facilitate future investigation s of normal and CML stem cell populations in vitro and in NOD/SCID mice.