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
Rm. Lemoli et Sc. Gulati, 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, Stem cells, 11(5), 1993, pp. 435-444
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.