FUNCTIONAL AND MORPHOLOGICAL CHARACTERIZATION OF IMMUNOMAGNETICALLY SELECTED CD34(+) HEMATOPOIETIC PROGENITOR CELLS

We evaluated the potential of immunomagnetically selected (miniMACS) p rogenitor cells to give rise to colony-forming cells and their precurs ors, detected as long-term culture-initiating cells (LTC-IC), as wed a s their capacity to expand in liquid cultures. A 90% mean purity, a 43 .2% yield and a 55.8-fold enrichment were achieved from normal bone ma rrow. When corrected for enrichment, the mean number of committed prog enitor cells and the frequency of LTC-IC (evaluated by means of limiti ng dilution assay [LDA]) were not statistically different in low densi ty mononuclear cells or in the CD34-enriched fractions, In five cases CD34(+) selected cells groan in a stroma-free long-term bone marrow cu lture system with the addition of stem cell factor, interleukin 3, int erleukin 6 and GM-CSF every 48 h, showed a 15 (+/-15) and 31 (+/-21) m ean colony forming unit-granulocyte/macrophage fold increase on cultur es at days 7 and 14. However, when corrected for enrichment, the expan sion capability of these cells was significantly lower than that of th e unseparated fraction, particularly after the first week. Immediately after separation, electron microscopy revealed that the CD34(+) selec ted fraction contained more than 45% of well-differentiated myeloid ce lls (MPO(+)), with iron beads preferentially clustered at one pole of the cell surface and sometimes already endocytosed in pinocytic vesicl es. After 24 h and 48 h incubation at 37 degrees C, the majority of th e cells shelved no iron particles, but about 30% of the cells were iro n-labeled phagocytic cells, The percentage of apoptotic cells with int ernalized iron was negligible. These data show that immunomagnetically separated CD34(+) cells may have a slightly impaired short-term expan sion capability, but give rise to both committed and more primitive pr ogenitor cells, During the separation, the iron beads are internalized , rapidly processed in the cytoplasm and do not seem to interfere with in vitro growth.