Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34(+) blood progenitor cells

So far, blood progenitor cells (BPC) expanded ex vivo in the absence of str omal cells have not been demonstrated to reconstitute hematopoiesis in myel oablated patients. To characterize the fate of early hematopoietic progenit or cells during ex vivo expansion in suspension culture, human CD34(+)-enri ched BPC were cultured in serum-free medium in the presence of FLT3 ligand (FL), stem cell factor (SCF) and interleukin 3 (IL-3). Both CD34 surface ex pression levels and the percentage of CD34(+) cells were continuously downr egulated during the culture period. We observed an expansion of colony-form ing units granulocyte-macrophage (CFU-GM) and BFU-E beginning on day 3 of c ulture, reaching an approximate 2-log increase by days 5 to 7. Limiting dil ution analysis of primitive in vitro clonogenic progenitors was performed t hrough a week 6 cobblestone-area-forming cell (CAFC) assay, which has previ ously been shown to detect long-term bone marrow culture-initiating cells ( LTC-IC). A maintenance or a slight (threefold) increase of week 6 CAFC/LTC- IC was found after one week of culture. To analyze the presence of BPC medi ating in vivo engraftment, expanded CD34(+) cells were transplanted into pr eirradiated NOD/SCID mice at various time points. Only CD34(+) cells cultur ed for up to four days successfully engrafted murine bone marrow with human cells expressing myeloid or lymphoid progenitor phenotypes. In contrast, f ive- and seven-day expanded human BPC did not detectably engraft NOD/SCID m ice. When FL, SCF and IL-3-supplemented cultures were performed for seven d ays on fibronectin-coated plastic, or when IL-3 was replaced by thrombopoie tin, colony forming cells and LTC-IC reached levels similar to those of con trol cultures, yet no human cell engraftment was recorded in the mice. Also , culture in U-bottom microplates resulting in locally increased CD34(+) ce ll density had no positive effect on engraftment. These results indicate th at during ex vivo expansion of human CD34(+) cells, CFC and LTC-IC numbers do not correlate with the potential to repopulate NOD/SCID mice. Our result s suggest that ex vivo expanded BPC should be cultured for limited time per iods only, in order to preserve bone-marrow-repopulating hematopoietic stem cells.