IN-VIVO AND IN-VITRO CHARACTERIZATION OF LONG-TERM REPOPULATING PRIMITIVE HEMATOPOIETIC-CELLS ISOLATED BY SEQUENTIAL HOECHST-33342-RHODAMINE-123 FACS SELECTION

Subpopulations of very primitive hematopoietic cells were isolated by fluorescence-activated cell sorter (FACS) selection of density gradien t-enriched, lineage-depleted marrow cells with blast cell light scatte r characteristics that bound low levels of the DNA binding dye, Hoechs t 33342 (Ho) and retained differential amounts of the mitochondrial bi nding dye, rhodamine 123 (Rh-123). The dyes were used sequentially in a single sorting operation. The subfractions of cells that stained mos t weakly with both dyes were highly coenriched for long-term repopulat ing cells (LTRC) and for in vitro high proliferative potential colony- forming cells (HPP-CFC). Furthermore, as populations of cells were pro gressively selected on the basis of decreasing Ho and Rh-123 fluoresce nce, first the CFU-S-8, then the CFU-S-12 diminished or disappeared en tirely in the lowest Rh-123 fraction. In these low fluorescent populat ions, plating efficiency for HPP-CFC was very high when cultured in th e combined presence of recombinant rat stem cell factor (rrSCF), recom binant human interleukin-l (rhIL-1), recombinant murine interleukin-3 (rmIL-3) and recombinant human colony-stimulating factor-1 (rhCSF-1), apparently reaching 100% in some instances. When 20 male donor cells f rom this lowest fluorescent Ho/Rh-123 fraction were injected into leth ally irradiated female recipients, along with a ''compromised'' marrow cell population (3x previously transplanted nonsorted female bone mar row cells), the sorted male donor cells were able to completely and ex clusively repopulate the myeloid and the lymphoid B and T cell compart ments of the recipients for at least 10 months posttransplant. Assays of cell fractions that were relatively more Rh-123 fluorescent demonst rated the presence of cells with progressively less repopulating capac ity. When descendants of transplanted low fluorescent Rh-123 selected cells, as found in 12-day spleen colonies, were assayed for the capaci ty to provide long-term survival in secondary recipients, they were ab le to do so in a high proportion of lethally irradiated recipients. Ho wever, spleen colonies derived from the mid-high fluorescence fraction were completely unable to do so. In summary we have demonstrated with a sequential Ho/Rh-123 sorting system that a subset of HPP-CFC cofrac tionate with LTRC with high frequency. Using this system, the enrichme nt of LTRC in the lowest Ph123 compartment of the sequentially Ho/Rh-1 23 selected cells appears to be the greatest demonstrated thus far. In addition, this study further supports previous ones that identify a c ompartment of LTRC that are largely distinct from CFU-S-12.