title: Cell separation improves the sensitivity of detecting rare human normal and leukemic hematopoietic cells in vivo in NOD/SCID mice.
abstract: 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.
  
  
  CONCLUSIONS
  This simple cell-depletion procedure should facilitate future investigations of normal and CML stem cell populations in vitro and in NOD/SCID mice.
authors.affiliation: Academic Transfusion Medicine Unit, Department of Medicine, Royal Infirmary, Glasgow, UK.
authors.forename: T L
authors.initials: TL
authors.lastname: Holyoake
authors.forename: C
authors.initials: C
authors.lastname: Horrocks
authors.forename: T
authors.initials: T
authors.lastname: Thomas
authors.forename: C J
authors.initials: CJ
authors.lastname: Eaves
authors.forename: A C
authors.initials: AC
authors.lastname: Eaves
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D002469:Cell Separation / Q000379:methods*; D005312:Fetal Blood / Q000166:cytology / Q000378:metabolism / Q000637:transplantation; D005434:Flow Cytometry; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D016130:Immunophenotyping; D007938:Leukemia / Q000235:genetics / Q000473:pathology*; D017493:Leukocyte Common Antigens / Q000032:analysis; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D018952:Antigens, CD34; D016044:Fusion Proteins, bcr-abl; D016315:Proto-Oncogene Proteins c-abl; D017493:Leukocyte Common Antigens