gp130-Stimulating designer cytokine Hyper-interleukin-6 synergizes with murine stroma for long-term survival of primitive human hematopoietic progenitor cells

Objective. Experimental strategies for ex vivo expansion of human hematopoi etic stem/progenitor cells involve the use of exogenous cytokines as well a s direct interaction with stromal elements. We examined the use of the inte rleukin-6/soluble interleukin-6 receptor (IL-6/sIL-6R) fusion protein Hyper -IL-6 (H-IL-6), which interacts directly with gp130, in conjunction with st romal support for expansion of human progenitors. Materials and Methods. Peripheral blood CD34(+) cells were cultured on the murine stromal cell line FBMD1 or in suspension for up to 28 days with diff erent cytokines, Cells were evaluated at various time points for phenotype, proliferative and clonogenic capacity, and longterm hematopoietic activity , Results. The combination of Flt3 ligand and H-IL-6 was markedly more effect ive than Flt3 ligand and IL-6/sIL-6R for expansion of CD34(+) cells in susp ension culture and on FBMD1. Addition of kit ligand but not thrombopoietin to Flt3 ligand and H-IL-6 significantly augmented proliferation and enhance d colony formation three-fold. However, long-term cobblestone area-forming cell assays indicated that although multipotent progenitors were maintained up to 21 days on FBMD1 in the presence of Flt3 ligand alone and were ampli fied three-fold by addition of H-IL-6, CD34+ cells cultured in the absence of stromal support rapidly lost their cobblestone area-forming cell potenti al. Immunophenotyping revealed that stromal support prevented upregulation of IL-6R on CD34(+) cells, which was induced within 3 days in stroma-free c ultures and was enhanced in the presence of kit ligand, Delayed addition of H-IL-6 to the cultures resulted in reduced proliferation and colony-formin g unit potential. Conclusion. H-IL-6 synergizes with stromal elements to effectively enhance proliferation and maintenance of primitive hematopoietic progenitors under prolonged ex vivo culture conditions. (C) 2001 International Society for Ex perimental Hematology. Published by Elsevier Science Inc.