Biological properties of subpopulations of pluripotent hematopoietic stem cells enriched by elutriation and flow cytometry.
We have studied several features of pluripotent hematopoietic stem cells (PHSCs) and day-12 spleen colony-forming units (CFU-S) obtained from adult murine bone marrow. Single-cell suspensions of C57BL/6J mouse bone marrow were fractionated by counterflow centrifugal elutriation at flow rates (FR) of 15, 25, 30, and 35 ml/min, and with the rotor off (R/O). The fractions FR25 and FR35 contained approximately equal numbers of PHSC that could repopulate W/Wv mice. These PHSCs were further enriched by subtracting lineage-positive cells using monoclonal antibodies (MAb) and magnetic immunobeads. The resulting lineage-negative cells (Lin-) were then stained with a MAb for the c-kit receptor and sorted by flow cytometry. Both subsets were fractionated into cells expressing high (bright) (c-kitBR), low (dull) c-kitDULL and no (negative, c-kitNEG) c-kit receptor. As few as 100 to 200 c-kitBR cells could repopulate the entire thymus and bone marrow in W/Wv mice. No PHSCs were present in the c-kitDULL and c-kitNEG fractions. We assayed fresh bone marrow and elutriation fractions FR25 and FR35 for gene expression by reverse transcriptase polymerase chain reaction. Using a semiquantitative protocol, we detected mRNA for beta-globin and flk-2, a protein tyrosine kinase receptor, in all samples except the FR25 Lin- c-kitBR subset. We consider the cells in FR25 Lin- c-kitBR to be the most primitive set of hematopoietic stem cells.
Orlic
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Clinical Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Anderson
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Bodine
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D000818:Animals; D002452:Cell Count; D002469:Cell Separation / Q000379:methods; D003114:Colony-Forming Units Assay; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis; D016184:Receptors, Colony-Stimulating Factor / Q000032:analysis; D020112:Rhodamine 123; D012235:Rhodamines; D013154:Spleen / Q000166:cytology*
D005456:Fluorescent Dyes; D011518:Proto-Oncogene Proteins; D016184:Receptors, Colony-Stimulating Factor; D012235:Rhodamines; D020112:Rhodamine 123; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases