ISOLATION OF PRIMITIVE HUMAN BONE-MARROW HEMATOPOIETIC PROGENITOR CELLS USING HOECHST-33342 AND RHODAMINE-123

In addition to possessing multilineage differentiation and self-renewa l capabilities, pluripotent hematopoietic stem cells are believed to b e mitotically quiescent and metabolically inactive. Fractions of human bone marrow (BM) CD34(+) cells can be further enriched for primitive hematopoietic progenitor cells (HPC) by using a number of cell-surface markers. All of these fractions, however, contain cells that are stil l heterogeneous as far as their metabolic and mitotic activities are c oncerned. We therefore used Hoechst 33342 (Hst) to identify quiescent cells and Rhodamine 123 (Rh123) to identify metabolically inactive cel ls. CD34(+)Hst(dim)Rh123(dim) (CD34(+)d/d) and CD34(+)Hst(bright)Rh123 (bright)(CD34(+)b/b) cells were isolated by flow cytometry to examine the hematopoietic functions of mitotically and metabolically homogeneo us progenitors. Cell-cycle status, progenitor cell content, maintenanc e of in vitro hematopoiesis, and long-term hematopoietic culture-initi ating cell (LTHC-IC) content of CD34(+)d/d and CD34(+)b/b cells were c ompared with CD34(+)HLA-DR(-) cells, a well-defined phenotype of primi tive HPC. Whereas 99.2 +/- 0.5% of freshly isolated CD34(+)d/d cells w ere in G(0)/G(1) phase of the cell cycle, only 74.4 +/- 11.5% of CD34( +)b/b and 75.6 +/- 1.1% of CD34(+)HLA-DR(-) cells were in G(0)/G(1). T he number of multipotential progenitors (colony-forming its-granulocyt e/erythroid/macrophage/megakaryocyte [CFU-GEMM]) detected in CD34(+)d/ d cells was twice that observed in CD34(+)HLA-DR(-) cells and eight ti mes that in CD34(+)b/b cells. In stromal cell-free long-term cultures maintained for 10 weeks, production of assayable progenitors in cultur es initiated with CD34(+)d/d cells exceeded that detected in CD34(+)HL A-DR(-) cultures by more than three-fold. Only in CD34(+)d/d cultures were high proliferative potential colony-forming cell (HPP-CFC)-derive d colonies detected over a period of 6 weeks. Limiting dilution analys is revealed that the frequency of LTHC-IC was highest among CD34(+)d/d cells (7.2 +/- 3.3%), followed by a frequency of 4.5 +/- 4.8% for CD3 4(+)HLA-DR(-) cells and 2.2 +/- 3.5% for CD34+b/b cells. The primitive nature of HPC identified by CD34, list, and Rh123 was confirmed by th e ability of as few as 200 murine marrow cells isolated by this techni que to radioprotect and fully reconstitute lethally irradiated recipie nts. These results indicate that Hst and Rh123 staining can be used in combination with CD34 immunofluorescence to isolate a quiescent subpo pulation of human primitive hematopoietic progenitor cells. Cells isol ated by this technique appear to have functional properties associated with stem cells, suggesting that they may be ideal candidates for stu dies requiring primitive HPC, such as ex vivo expansion and somatic ge ne therapy.