A fractionation procedure of mouse bone marrow cells yielding exclusively pluripotent stem cells and committed progenitors

The cell surface phenotype of pluripotent hemopoietic stem cells (CFU‐S) and committed progenitors (CFU‐C1, CFU‐C2, BFU‐E) of mouse bone marrow was analyzed with respect to their binding of wheat germ agglutinin (WGA) and two monoclonal antibodies, anti‐GM‐1.2 and anti‐PGP‐1. Stained cells were fractionated on the basis of differences in fluorescence and light scatter intensity using a light‐activated cell sorter. The 6% of the cells that bound most WGA and that also had a relatively high forward light scatter (FLS) and low perpendicular light scatter (PLS) contained nearly all stem cells (CFU‐S) and progenitors. Anti‐GM‐1.2 stained only mature myeloid cells, not CFU‐S or the in vitro colony‐forming cells. Anti‐PGP‐1 stained all bone marrow cells in varying intensities: lymphoid cells were dull, CFU‐S were intermediate, CFU‐C2 were brighter, and mature myeloid cells very bright. Enrichment of progenitor cells was performed by a two‐step sorting procedure. First, the 6% most WGA‐binding cells with high FLS and low PLS were sorted out. A 10–15‐fold enrichment of progenitors and CFU‐S was obtained. Next, these cells were restained with anti‐GM‐1.2 or anti‐PGP‐1 and again fractionated on the FACS. The GM‐1.2‐negative cells were then another four‐ to sevenfold more enriched for stem cells and progenitors. Of the cells in this fraction, 95% could be assigned to a colony‐forming unit. With anti‐PGP‐1, CFU‐C2 could be partly separated from more early cells such as CFU‐S and BFU‐E.