Further Studies of Galectin-1 (Gal-1) on CD34⁺ Umbilical Cord Blood Cells.

The galectins are a family of carbohydrate-binding proteins that are distributed widely in vertebrate organisms. Each galectin exhibits a specific pattern of expression in various cells and tissues. Although these proteins are found mainly in the cytoplasm, some of them are secreted from cells and interact with other glycosylated molecules at the cell surface or with the extracellular matrix. There is no information about the interaction of Gal-1 and hematopoietic progenitor cells (HPC). We studied the effect of rat recombinant galectin-1 (rGal-1) on the clonogenic capability and the morphologic differentiation of the human umbilical cord blood HPC in methylcellulose cultures. The quantification of total anti-Gal-1 binding capability (total receptors) was also determined by flow citometry. First, the cells of 10 human cord blood units were collected and CD34⁺ selected using an indirect immunomagnetic method (MidiMACS, Miltenyi, Biotec). Ten thousand events were acquired in a flow cytometer (FACScan, Becton Dickinson), employing 10⁵ CD34⁺ cells, rabbit anti Gal-1 antiserum and anti-rabbit IgG FITC antibody. Finally the CD34⁺ cells were cultured in a semisolid methylcellulose medium (Methocult GF⁺ H4435, StemCell) by triplicate at 37°C, 5% CO₂ and 100% humidity during 14 days, employing 24 wells culture plates (Nunc) in presence or not of different concentrations of rGal-1 (2, 4, 6 and 20 μg/ml). We analyzed the number of GEMM-CFU (mix colonies), GM-CFU (granulocyte- macrophage colonies), BFU-E (erythroid colonies) and the presence of signals of abnormal morphologic differentiation. Control of inhibition with thiodigalactoside (TDG) and lactose (Lac) 100 mM was done in every case to determine the assay specificity. Histograms show a total receptor mean expression (mean+/−SEM) in CD34⁺ cells of 18.2+/−2.44% which was significantly inhibited (p<0.05) with TDG (8.8+/−0.98%) and Lac (13.5+/−3.70%). A statistically significant increase in the number of GEMM-CFU, GM-CFU and BFU-E was seen. These increases were directly proportional to the rGal-1 concentration added (p<0.05) and it was stronger for GM-CFU. This effect was partially inhibited by TDG and Lac. We observed an abnormal increase in the mesenchymal, fibroblastic-like cell growth in methylcellulose cultures. These results showed the expression of Gal-1 in CD34⁺ human umbilical blood cells membranes. The addition of rGal-1 to cultures would increase the proliferation of HPC and the differentiation to stromal-like cells would partially occur. The mechanism and functional meaning of this activation and differentiation must still been elucidated.