NERVE GROWTH-FACTOR IS INVOLVED IN THE SUPPORTIVE EFFECT BY BONE-MARROW-DERIVED STROMAL CELLS OF THE FACTOR-DEPENDENT HUMAN CELL-LINE UT-7

We previously demonstrated that murine MS-5 and Sl/Sl(4) cell lines in duce the proliferation of human factor-dependent UT-7 cells in the abs ence of normally required human cytokines and also stimulate the diffe rentiation of CD34(+)/ CD38(-)LTC-ICs. We report in this study that th e effect of MS-5 cells on UT-7 cells can be completely explained by th e synergistic action of nerve growth factor (NGF) and stem cell factor (SCF) produced by these murine stromal cells. Purified murine NGF was able to support short-term clone formation and long-term growth of UT -7 cells in suspension cultures as efficiently as rhu-granulocyte-macr ophage colony-stimulating factor. NGF action was mediated through the TrkA receptor, in which messenger RNA (mRNA) was easily detected in UT -7 cells by Northern blot. MS-5 cells strongly expressed NGF mRNA in N orthern blot, and direct implication of MS-5-derived NGF in the induct ion of UT-7 cells proliferation was demonstrated in inhibition assays with an anti-NGF monoclonal antibody (MoAb) that neutralized by 84% +/ - 4.1% (n = 5) UT-7 clone formation. However, NGF did not act alone, a nd several arguments demonstrated the synergistic action of MS-5-deriv ed SCF: (1) an anti-c-kit partially inhibited UT-7 cells clone formati on in coculture assays, (2) SCF and NGF synergized in an H-3-TdR incor poration assay, and (3) the stimulatory effect of 10x-concentrated MS- 5 supernatant was completely inhibited by an anti-c-kit but not by an anti-NGF, and levels of soluble NGF (1.2 ng/mL) detected by enzyme-lin ked immunosorbent assay in 10x supernatant of MS-5 cells cultures were below the biologically active concentrations. In contrast, although M S-5 cells also promoted the differentiation of very primitive CD34(+)/ CD38(-) human stem cells both in colony assays and long-term cultures, we could not incriminate MS-5-derived NGF in the observed effect: an anti-NGF MoAb did not inhibit the synergistic effect of MS-5 cells in colony assays or longterm cultures nor did soluble muNGF duplicate MS- 5 effect and survival of CD34(+)/CD38(-) clonogenic progenitor cells p romoted by MS-5 was unaffected by an anti-NGF and was not induced by s oluble NGF alone or combined with SCF. In contrast, NGF in synergy wit h SCF supported the short-term maintenance of high numbers of CD34(+)/ CD38(+) mature erythroid progenitors probably through an indirect mech anism implying macrophages. These results suggest that NGF, in which t he primary target cells are outside the hematopoietic system, is prese nt in the marrow environment and might act at some steps of hematopoie tic stem cell development, These results also underline that the respo nse of cell lines and normal stem cells to stromal cells is mediated b y different pathways. (C) 1996 by The American Society of Hematology.