Purification of murine hemopoietic stem cells and committed progenitors by fluorescence activated cell sorting using wheat germ agglutinin and monoclonal antibodies

Abstract

Two procedures to purify and separate pluripotent hemopoietic stem cells (PHSC) and committed progenitor cells from mouse bone marrow cells, and a three colour stem cell staining procedure are described. Visser et al., (J. Exp. Med. 59, 1576-1590, 1984) described the purification of PHSC by metrizamide density gradient centrifugation followed by wheat germ agglutinin-FITC (WGA-FITC) and light scatter sorting using a fluorescence activated cell sorter (FACS). The light density, WGA-positive, high forward (FLS) and low perpendicular light scatter (PLS) blast cells, after removal of the lectin from the sorted cells by the competing sugar, are restained with biotinylated anti-H-2K plus avidin-FITC. The H-2K positive cells proved to be pure pluripotent stem cells. Bauman et al. (J. Cell. Physiol. 128, 133-142, 1986) started by sorting the 6% most positive fluorescent cells with low PLS and high FLS from WGA-FITC stained normal bone marrow. After lectin removal the sorted cells are restained with anti-GM-1.2. Sorted, GM-1.2 negative cells are almost exclusively PHSC plus committed colony forming cells. A three colour staining procedure was designed to measure stem cells in bone marrow samples by flow cytometry. Cells are simultaneously stained with anti-H-2K-biotin plus avidin-phycoerythrin, a rat monoclonal antibody detecting a cell surface antigen plus goat anti rat Ig-FITC and 1-butyryl-pyrene-WGA. Analysis and sorting on the two laser Rijswijk Experimental Light-Activated Cell Sorter (RELACS II) using selected windows indicated that these windows contained high frequencies of PHSC. This multicolour analysis and sorting therefore is equivalent to the multistep sorting procedures.