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Exp Hematol. 1993 May;21(5):614-22.

In vivo and in vitro characterization of long-term repopulating primitive hematopoietic cells isolated by sequential Hoechst 33342-rhodamine 123 FACS selection.

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  • 1Department of Pathology, University of Washington, Seattle 98195.


Subpopulations of very primitive hematopoietic cells were isolated by fluorescence-activated cell sorter (FACS) selection of density gradient-enriched, lineage-depleted marrow cells with blast cell light scatter characteristics that bound low levels of the DNA binding dye, Hoechst 33342 (Hö) and retained differential amounts of the mitochondrial binding dye, rhodamine 123 (Rh-123). The dyes were used sequentially in a single sorting operation. The subfractions of cells that stained most weakly with both dyes were highly coenriched for long-term repopulating cells (LTRC) and for in vitro high proliferative potential colony-forming cells (HPP-CFC). Furthermore, as populations of cells were progressively selected on the basis of decreasing Hö and Rh-123 fluorescence, first the CFU-S-8, then the CFU-S-12 diminished or disappeared entirely in the lowest Rh-123 fraction. In these low fluorescent populations, plating efficiency for HPP-CFC was very high when cultured in the combined presence of recombinant rat stem cell factor (rrSCF), recombinant human interleukin-1 (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 from this lowest fluorescent Hö/Rh-123 fraction were injected into lethally irradiated female recipients, along with a "compromised" marrow cell population (3x previously transplanted nonsorted female bone marrow cells), the sorted male donor cells were able to completely and exclusively repopulate the myeloid and the lymphoid B and T cell compartments of the recipients for at least 10 months posttransplant. Assays of cell fractions that were relatively more Rh-123 fluorescent demonstrated the presence of cell with progressively less repopulating capacity. When descendants of transplanted low fluorescent Rh-123 selected cells, as found in 12-day spleen colonies, were assayed for the capacity to provide long-term survival in secondary recipients, they were able to do so in a high proportion of lethally irradiated recipients. However, spleen colonies derived from the mid-high fluorescence fraction were completely unable to do so. In summary, we have demonstrated with a sequential Hö/Rh-123 sorting system that a subset of HPP-CFC cofractionate with LTRC with high frequency. Using this system, the enrichment of LTRC in the lowest Rh-123 compartment of the sequentially Hö/Rh-123 selected cells appears to be the greatest demonstrated thus far. In addition, this study further supports previous ones that identify a compartment of LTRC that are largely distinct from CFU-S-12.

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