LONG-TERM HEMATOPOIETIC CULTURE-INITIATING CELLS ARE MORE ABUNDANT INMOBILIZED PERIPHERAL-BLOOD GRAFTS THAN IN BONE-MARROW BUT HAVE A MORELIMITED EX-VIVO EXPANSION POTENTIAL

Mobilized peripheral blood hematopoietic progenitor cells obtained fro m cancer patients treated with high-dose cyclophosphamide (7g/m(2)) fo llowed by G-CSF, GM-CSF, IL-3, PIXY321, or combinations of these cytok ines have been successfully used for autologous stem cell transplantat ion. We investigated the ability of hematopoietic progenitor cells (HP C) derived from mobilized peripheral blood (PB) to undergo ex vivo exp ansion in short term cultures by enumerating numbers of de novo genera ted CD34(+) cells, assayable progenitor cells, and the frequency of lo ng-term hematopoietic culture-initiating cells (LTHC-IC), These parame ters were examined in CD34(+) cells generated in culture through the u se of cell tracking with the membrane dye PKH2. Fresh isolated mobiliz ed CD34(+) cells contained 0.49 - 0.36% LTHC-IC, However, due to the h igh number of total CD34(+) cells in mobilized PB, the absolute number of LTHC-IC was higher than that contained in a bone marrow (BM) harve st, Mobilized CD34(+) cells were stained with PKH2 and incubated with SCF, IL-3, and IL-6. After 5 to 6 days, numbers of total CD34(+) cells and clonogenic progenitors increased 1.4- and 2.2-fold, respectively. Numbers of total progenitors continued to increase such that 10 to 12 days after the initiation of cultures a 6.4-fold increase was demonst rable. However, between days 5 and 7 of culture, the frequency of LTHC -IC in CD34(+)PKH2(bright) cells (cells which did not divide) was less than 50% of that determined for fresh cells, while the frequency amon g CD34(+)PKH2(dim) cells (cells that had divided) was very low or unde tectable. However, moderately higher frequencies of LTHC-IC were detec ted following expansion for 48 hours only. In similar assays, both BM and cord blood cells were capable of generating LTHC-IC in CD34(+)PKH2 (dim) cells but not to expand the overall number of these progenitors, These observations suggest that although mobilized PB CD34(+) cells c ontain large numbers of LTHC-IC, these cells might not be capable of f urther ex vivo expansion and generation of additional LTHC-IC in vitro . Furthermore, these data indicate that mobilized PB CD34(+) cells may have undergone maximal ''in vivo expansion'' such that additional ex vivo expansion of primitive progenitor cells may not be possible.