Culture media were changed every 4 to 6 days. FISH analysis We cultured BCR/ABL+ hemangioblasts from male CML patients (n = 12) and Y chromosome was detected using a probe (CEP Y Spectrum Red; Vysis, Downers Apoptosis inhibitor Grove, IL) according to the manufacturer’s instructions. Normal cells showed 2 red abl signals and 2 green bcr signals. BCR/ABL+ hemangioblasts showed a single
red and a single green signal representing normal abl and bcr genes and the yellow signal representing fusion of abl and bcr genes. Fluorescence activated cell sorting (FACS) For immunophenotype analysis, expanded clonal cells were stained with antibodies against Flk1, CD29, CD31, CD34, CD44, CD45, CD105, (all from Becton Dickinson Immunocytometry Systems, Mountain View, CA). For intracellular antigen detection, cells were first fixed in 2% paraformaldehyde (Sigma) for 15 minutes at 4°C and permeabilized with 0.1% saponin (Sigma) for 1 hour at room temperature. Cells were washed
and labeled with fluorescein isothiocyanate (FITC) conjugated secondary goat antimouse, goat antirabbit, or sheep antigoat antibodies (Sigma), then washed and analyzed using a FACS Calibur flow cytometer (Becton VEGFR inhibitor Dickinson, San Jose, CA). Mitogen proliferative assays Inmitogen proliferative assays, triplicate wells containing responder 1 × 105 MNCs were cultured with 50 g/ml PHA (Roche, USA) in a total
volume of 0.1 ml medium at 37°C in 5% CO2, and Flk1+CD31-CD34- MSCs were added on day 0. Irradiated Flk1+CD31-CD34- MSCs (30 Gy) were cocultured with the MNCs at different ratios (MSCs to MNCs = 1:2, 1:10, 1:100). Control wells contained only MNCs. Cultures were pulsed with 1 Ci/well [3H]-TdR (Shanghai Nucleus Research Institute, China) on day 2, and harvested 18 h laterwith a Tomtec (Wallac Inc., Gaithersburg, Org 27569 MD) automated harvester. Thymidine uptake was quantified using a liquid scintillation and luminescence counter (Wallac selleck TRILUX). Mixed lymphocyte reaction assays (MLR) Blood mononuclear cells (MNCs) were prepared from normal volunteers’ peripheral blood by Ficoll-Paque density gradient centrifugation and suspended inRPMI 1640 medium supplemented with 10% (vol/vol) FCS, 2 mM l-glutamine,0.1 mM nonessential amino acids (Life Technologies, Grand Island, NY), 1 mM sodium pyruvate, 100 U/mL penicillin, Effect of MSCs on T cell cycle MSCs and MNCs were prepared as described before.