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

Abstract

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.