Effects of FGFR/Fc chimeras on stromal cell secretion of SDF-1 and supportive function for hematopoietic progenitor cells. (A) FGFR/Fc chimeras (0-2000 ng/mL in αMEM containing 10% FBS) were incubated in medium only or with FGF2 (10 ng/mL) for 30 minutes, and then added to stromal cell (MS-5 and S-17) monolayers (approximately 90% confluent). A specific anti–FGFR2 IIIc neutralizing mAb (0-2000 ng/mL) was first applied onto stromal monolayers for 30 minutes, and then FGF2 (10 ng/mL) was added. After 72-hour incubation, culture supernatants were collected and used to measure SDF-1 content by a specific SDF-1 ELISA. The percent SDF-1 secretion was calculated as follows: ([SDF-1 secretion in FGFR/Fc or anti–FGFR2 IIIc mAb group]/[SDF-1 secretion in control group with no additive]) × 100 (%). All experiments were performed at least in triplicate. Representative results from 4 independent experiments are shown. (B) MS-5 cells and S-17 cells (90% confluent, in 200 μL, 96-well plates) were incubated in medium alone, with FGFR/Fc chimeras, or with anti–FGFR2 IIIc mAb (2000 ng/mL) for 72 hours. The SDF-1 content was determined by a specific ELISA, and the results were expressed as percent SDF-1 secretion. All experiments were performed at least in triplicate and repeated 4 times. The results reflect the means (± SD) of 4 independent experiments. (C) MS-5 cells and S-17 cells were detached and washed twice with PBS. Cells were suspended in culture medium (αMEM containing 10% FBS), plated in triplicate (2000 cells/well in 0.2 mL culture medium) with the addition of FGFR/chimeras (2000 ng/mL) or with anti–FGFR2 IIIc mAb (2000 ng/mL) onto 96-well plates, and incubated for 64 hours. DNA synthesis was measured by ³ H thymidine deoxyribose uptake during the last 16 hours of culture. The results are expressed as percent ³ H incorporation. All experiments were performed at least in triplicate and repeated 4 times. The results reflect the means (± SD) of 4 independent experiments. (D) MS-5 cells (80%-90% confluent on gelatin-coated 24-well plates) were incubated in medium alone, with FGFR1 IIIc/Fc chimera (800 ng/mL), with FGF2 (10 ng/mL), with FGFR1 IIIc/Fc chimera (800 ng/mL) plus FGF2 (10 ng/mL), or FGFR1 IIIb (800 ng/mL) plus FGF2 (10 ng/mL) for 72 hours, and then washed twice with PBS. Human CD34⁺ peripheral blood progenitor cells (PBSCs: 5 × 10⁴ cells in 0.36 mL) in medium alone, with FGFR1 IIIc/Fc chimera (800 ng/mL), with FGF2 (10 ng/mL), with FGFR1 IIIc/Fc chimera (800 ng/mL) plus FGF2 (10 ng/mL), or FGFR1 IIIb/chimera (800 ng/mL) plus FGF2 (10 ng/mL) were applied onto pretreated stromal layers (4 wells/subgroup). Cocultures were incubated for 3 weeks with replenishment of culture medium alone (0.18 mL) or with the appropriate original additives (800 ng/mL FGFR1 IIIc, 10 ng/mL FGF2, 800 ng/mL FGFR1 IIIc plus 10 ng/mL FGF2, or 800 ng/mL FGFR1 IIIb plus 10 ng/mL FGF2) twice/wk. After 3 weeks, nonadherent viable cells were counted and analyzed by FACS. The percent growth of PBSCs was calculated as ([CD45⁺ cell number with FGFR1 IIIc/Fc, FGF2, FGF2 plus FGFR1 IIIc/Fc, or FGF2 plus FGFR1 IIIb/Fc]/[CD45⁺ cell number with no additive]) × 100 (%). The results reflect the means (± SD) of 3 independent experiments. The asterisk denotes statistical significance (**P < .01; *P < .05).