Negative influence of IL3 on the expansion of human cord blood in vivo long-term repopulating stem cells

Abstract

Identification of culture conditions that support expansion or even long-term maintenance of in vivo repopulating human hematopoietic stem cells is still a major challenge. Using a combination of FLT3 ligand (FL), Stem Cell Factor (SCF), Thrombopoietin (TPO) and Interleukin 6 (IL6), we cultured cord blood (CB) CD34+ cells for up to 12 weeks and transplanted their progeny into sublethally irradiated NOD/SCID mice. Bone marrow engraftment was considered successful when recipients contained measurable numbers of human CD45+, CD71+ and Glycophorin A+(GpA) cells 8 weeks after transplantation. Twelve-week expanded cells with FL+SCF+TPO+IL6 successfully engrafted all of the recipients and human CD45(+)+CD71(+)+GpA(+) cells represented 4.3 to 22.4% of bone marrow. Substitution of IL6 with IL3 led to an even better expansion of cells and a similar clonogenic progenitor output in the first 8 weeks of culture; however, LTC-IC output increased up to week 6 and then decreased and disappeared. By contrast, with FL+SCF+TPO+IL6, LTC-IC kept increasing up to week 12. Four-week cultured cells with FL+SCF+TPO+IL3 less efficiently engrafted NOD/SCID mice, both as measured by frequency of positive recipients (4 out of 10) and percentage of engrafted human cells (< or =2%). Six-week expanded cells failed to engraft. This study provides evidence that many, but not all, of the so-called "early acting" cytokines, can sustain long-term maintenance and even expansion of human primitive in vivo repopulating stem cells. In particular, in the culture conditions used in this study, the presence of IL3 greatly reduces the repopulating potential of expanded CD34+ CB cells.