Primitive hematopoietic progenitor cells are present in peripheral blood autografts

In this report we have used an in vitro assay for long-term culture-initiating cells (LTC-IC) to detect primitive hematopoietic progenitor cells (HPC) in the peripheral blood (PB) of cancer patients who received high-dose cyclophosphamide (HD-CTX) followed by a combination of recombinant hematopoietic growth factors (C-HGF) including either interleukin-3 (IL-3)+ granulocyte colony-stimulating factor (G-CSF), IL-3+ granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3/GM-CSF fusion protein (PIXY-321). In addition, we have developed a quantitative assay for cells capable of generating additional colony-forming cells (pre-CFC) as a means of determining primitive HPC present in mobilized PB cells. CD34⁺ human leukocyte A (HLA)-DR^- cells isolated from the mobilized PB were capable of initiating long-term hematopoiesis in vitro that persisted for 10 weeks, while CD34⁺ HLA-DR^- cells obtained from the nonmobilized PB or BM were capable of sustaining long-term hematopoiesis in vitro for only 4 weeks and 8 weeks, respectively. As determined by a limiting dilution analysis of mobilized PB CD34⁺ HLA-DR^- cells, the frequency of pre-CFC was 4.3% (range, 1.0-8.3%) Pre-CFC comprised 0.01% (range, 0.001-0.02%) of mobilized PB mononuclear cells, and 151 pre-CFC were calculated to be present in one milliliter of mobilized PB (range, 20-310/ml). These results suggest that PB mononuclear cells collected by leukaphoresis following mobilization with HD-CTX + C-HGFs contain not only differentiated HPCs but also more primitive HPC.