Stem cell factor (scf)

Either sex of ICR mice aged 4-7 days were sedated with diethyl ether and killed by cervical dislocation. Small intestine (from 1 cm below the pyloric ring to the cecum) and colon (from below cecum to rectum) were cut and pinned to the base of sylgard dish full with ice-cold Ca²⁺ free Hank’s solution (see solutions). Tissues were opened along the mesenteric border. After removing luminal contents, the mucosa and submucosa of tissues were peeled away by sharp dissection. Strips of intestinal or colonic muscle were equilibrated in Ca²⁺ free Hank’s solution for 30 minutes. The muscle strips were transferred into enzymic solution (see solutions) and incubated in a 37℃ water bath for 14 minutes. Then tissues were washed out 3 times with Ca²⁺ free solution and triturated with blunt pipettes to disperse cell lumps. Cells were plated on poly-L-lysine (200 μL; Sigma, St. Louis, MO, USA) coated sterile glass coverslips in 35 mm culture dishes and incubated at 37℃ in a 95% O₂-5% CO₂ incubator in smooth muscle growth medium (SMGM; Lonza, Walkersville, MD, USA) supplemented with 2% antibiotic-antimycotics (Gibco, Grand Island, NY, USA) and stem cell factor (5 ng/mL; Sigma,St. Louis, MO, USA). After 1 day incubation, the medium was replaced with SMGM without stem cell factor, then incubated further for 24 hours with the same conditions until performing the following experiments.

The Kasumi-1 cells were purchased from ATCC and were maintained in RPMI 1640 (Millipore, Merck) medium supplemented with 10% fetal bovine serum (Sigma), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37°C in humidified air with 5% CO₂.
The bone marrow CD34+ cells were purchased from ATCC and thawed using IMDM containing 10% FBS and 20 U/mL Deoxyribonuclease I (Sigma) and grown in Iscove’s Modified Dulbecco’s Medium (IMDM, Lonza) supplemented with 15% fetal bovine serum, 1% penicillin-streptomycin, 10 μg (25 ng/mL) stem cell factor (Sigma), 4 μg (10 ng/mL) In-6 and In-3 (Sigma). The cells were cultured in a 5% carbon dioxide humidified atmosphere at 37°C.

After informed consent was obtained, 20 mL of peripheral blood was withdrawn from 30 patients before HU treatment. Primary cell cultures were performed as previously described.^{25 (link)} Mononuclear cells from peripheral blood were isolated by centrifugation over Ficoll-Hypaque (1.077 g/mL; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) at 1200 × g for 15 min. The nucleated cells were first cultured in a minimal essential medium (MEM) supplemented with 1 μg/mL cyclosporine A (Novartis Basilea, Switzerland), 10% fetal calf serum (FCS, Invitrogen, Carlsbad, California, USA) and 10% conditioned medium collected from cultures of the human bladder carcinoma 5637 cell line. After 6 days of incubation in phase I culture, the non-adherent cells were harvested, washed, and resuspended in phase II medium composed of α-MEM, 30% FCS, 1% deionized bovine serum albumin (Sigma-Aldrich, St. Louis, Missouri, USA), 10 mM 2- mercapto-ethanol (Sigma), 1.5 mM glutamine (Euroclone), 1 mM dexamethasone (Laboratorio Farmacologico Milanese, MI, Italy), 1U/mL recombinant erythropoietin (Janssen-Cilag, Leiden, The Netherlands), 10 ng/mL Stem Cell Factor (Sigma), and 0.3 mg/mL human holo-transferrin (Sigma). Cells were harvested at day 10 of phase II culture. We performed at least two different primary cultures for each patient.