EFFECT OF STEM-CELL FACTOR (C-KIT LIGAND), GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR AND INTERLEUKIN-3 ON HEMATOPOIETIC PROGENITORS IN HUMAN LONG-TERM BONE-MARROW CULTURES

In this paper we attempt to improve upon the methods of hematopoietic stem cell expansion. We evaluate the effects of recombinant human stem cell factor (SCF or c-kit ligand) alone and also in combination with recombinant human granulocyte-macrophage colony stimulating factor (GM -CSF) and interleukin 3 (IL-3), on cell proliferation and differentiat ion in human long term bone marrow cultures (LTBMC). Weekly addition o f 5 ng/ml of SCF with 25% serum containing media resulted in increased recovery of total nonadherent cells, granulocyte-macrophage colony fo rming units (CFU-GM), and burst-forming units erythroid (BFU-E) at wee k 1, but the number of bone marrow (BM) progenitor cells fell below th e level of untreated control cultures at weeks 3 (BFU-E) and 4 (CFU-GM ). At week 8, when the cultures were terminated, the CFU-GM recovery w as markedly reduced in flasks supplemented with SCF compared with the controls (p < 0.002). Moreover, SCF treatment induced the early disapp earance of BFU-E. When LTBMC were supplemented with the combination of SCF plus GM-CSF (100 U/ml) and IL-3 (5 ng/ml), synergistic activity o f the CSFs was observed at week 1. The number of BM colony forming cel ls (CFC) rapidly declined below the level of growth factor-free contro ls, leading to the early exhaustion of the culture when SCF was combin ed with GM-CSF. By comparison, GM-CSF and IL-3 alone induced a statist ically significant increase above the controls (no growth factor) in t he number or nonadherent cell colonies of CFU-GM and BFU-E. Analysis o f adherent layer cells from cultures supplemented with SCF showed incr eased cellularity, no adipogenesis, and early disappearance of myeloid progenitors while the percentage of CFU-GM in S phase, assessed by cy tosine arabinoside (Ara-C) suicide assay, was 9.2 +/- 5% SD versus 27. 7 +/- 10% SD in control (no growth factor) samples (p < 0.01). SCF inc reased the number of fibroblast colony forming units (CFU-F) and also showed a synergistic activity (9.6-fold increase) when combined with I L-3. These findings suggest that SCF, GM-CSF and IL-3 exert their acti vity on different cell populations within the hematopoietic system. Fu rther investigations are needed to optimize the use of SCF in supporti ng hematopoiesis.