Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Although long-term repopulating hematopoietic stem cells (HSC) can self-ren ew and expand extensively in vivo, most efforts at expanding HSC in vitro h ave proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal com bination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-ll have emerged as likely p ositive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of Introduction HSC fate by different cytokine s, However, the interpretations of these findings are complicated by the fa ct that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewa l. Here, in the presence of KL+FL+megakaryocyte growth and development fact or (MGDF), which recruits virtually all Lin(-)Sca-1(+)kit(+) bone marrow ce lls into proliferation and promotes their self-renewal under serum-free con ditions, IL-3 and IL-ll revealed an indistinguishable ability to further en hance proliferation. Surprisingly, and similar to IL-ll, IL-3 supported KLFL+MGDF-induced expansion of multilineage, long-term reconstituting activit y in primary and secondary recipients. Furthermore, high-resolution cell di vision tracking demonstrated that all HSC underwent a minimum of 5 cell div isions, suggesting that long-term repopulating HSC are not compromised by I L-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulte d in extensive loss of reconstituting activity, an effect further facilitat ed by the presence of IL-3. (C) 2000 by The American Society of Hematology.