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.