Cytokine-mediated ex vivo expansion has been proposed as a means of increasing the number of cord blood (CB) hematopoietic stem cells for transplantation. As well as stem cell number, stromal cells are necessary for functional maturation of hematopoiesis. The purpose of this study was to analyze the development of stromal cells during ex vivo expansion of CB CD34⁺ cells.

CD34⁺ cells were purified from CB by magnetic bead selection. The levels of of interleukin-3, interleukin-1β, interleukin-6, granulocyte macrophagecolony stimulating factor and tumor necrosis factor-α were measured in culture supernatants on 0, 1, 2, and 3 weeks, using ELISA techniques. CB CD34⁺ cells were expanded in Iscoves modified Dulbeccos medium in the presence of several cytokines. The expression of E-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, platelet/endothelial cell adhesion molecule-1, von Willebrand factor, vimentin, and CD14 in newly developed stromal cells was examined by immunocytochemical method. Relevant extracellular matrix (ECM) proteins and proper cytokines were also assayed for the most suitable condition for expansion of stromal cells.

Several cytokines were found to have been produced by CB CD34⁺ cells as well as bone marrow-derived CD34⁺ cells. During ex vivo expansion of CB CD34⁺ cells, stromal cells appeared in the culture by day 4 and expanded over the following 7-10 days before being confluent by day 2 1. These cells expressed surface markers characteristic of cells of endothelial lineage. Furthermore, these stroaml cells also expanded effectively when treated with thrombopoietin+flt-3 ligand+stem cell factor+leukemia inhibitory factor or 0.1% poly-L-lysine-coated wells.

Stromal cells were developed during ex vivo expansion of CB CD34⁺ cells and that this development could be enhanced further by treating the stromal cells with cytokines or ECM.