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 improve 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 cells were labeled with a combination of Abs specific for murine hematopoietic cells, prior to immunomagnetic negative selection using StemSep. In subsequent experiments, BM was harvested from individual NOD/SCID mice transplanted 6-12 weeks earlier with either human cord blood or primary CML cells and a similar immunomagnetic selection procedure was applied to enrich human cells present. RESULTS: Application of this selection procedure to mixtures of murine and human hematopoietic cells using anti-mouse CD45 and Ter-119 allowed a > 1000-fold depletion of murine cells with > 50% recovery of human cells, including progenitors. This level of depletion and recovery were found to be reproducible for NOD/SCID mice transplanted and engrafted with human cord blood stem cells, thus facilitating detection of human progenitors, including colony-forming cells (CFC) and LTCIC. For NOD/SCID mice previously transplanted with CML cells, this procedure increased the sensitivity of detecting rare human cell subsets by up to > 100-fold. This, in turn, improved the sensitivity of RT-PCR for BCR-ABL and made possible the identification by FACS of various minor subsets of human cells, including CD34(-)CD19/20(+) B-lineage cells, CD34(+) progenitors, mature CD15(+) myeloid cells and CD3(+) T cells present in the mice. DISCUSSION: This simple cell-depletion procedure should facilitate future investigations of normal and CML stem cell populations in vitro and in NOD/SCID mice.