Ex Vivo Expansion of Stem Cells: Defining Optimum Conditions Using Various Cytokines

Abstract

With the increasing information on the number, quality, and characteristics of hematopoietic stem cells (HSC) in umbilical cord and placental blood, this material has been found to be efficacious as an alternative source of HSC for transplantation in children. In this study, we sought to define the optimal conditions for ex vivo expansion of cord blood (CB) stem cells. These conditions include: the combinations and concentrations of hematopoietic growth factors (stem cell factor [SCF], granulocyte-macrophage colony-stimulating factor [GM-CSF], interleukin [IL]-3, thrombopoietin [Tpo], IL-6 and Fms-like tyrosine kinase 3 ligand [Flt-3L]), the duration of culture, and the effect of serum supplementation. In this study, 2 protocols were applied for ex vivo expansion of CB stem cells. In protocol I, 20 CB samples were expanded in a static, serum-added, liquid culture for 7 and 11 days using 5 cytokine cocktails. In protocol II, 10 CB samples were expanded for 7 days using cytokines of cocktail 1, with and without IL-6 and Flt-3L, in serum-added and serum-free culture media. This protocol was intended to verify the effect of IL-6, Flt-3L, and the role of serum supplementation in short-term liquid culture. From the present study, it can be concluded that cocktail 1 is the cocktail of choice for ex vivo expansion of CB stem cells in serum-free, liquid culture expanded for 7 days. We can also conclude that culture expanded for 7 days is better than 11 days, as the fold expansion of CD34+ cells was not significantly increased or even decreased in some of the cocktails used. Moreover, the percent of CD95+ cells (apoptotic cells) was significantly increased on day 11 compared to day 7 in the cocktails tested.