Expansion of megakaryocyte progenitors from human umbilical cord blood using a new two-step separation procedure

Cord blood (CB) transplantation is primarily performed in children, rather than in adults, due to the low number of haemopoietic progenitor cells obtained from the small volume of a single CB collection. Prolonged thrombocytopenia is a major problem following CB transplantation. Efforts are currently underway to expand the number of CB progenitor cells ex vivo, in order to enable transplantation in adults and to decrease the period of thrombocytopenia. In this study we investigated different techniques for enrichment and expansion of megakaryocyte (Mk) progenitor cells and haemopoietic stem cells from CB. CBs from 20 normal deliveries were depleted of red blood cells (RBC) by dividing each sample and testing cell separation on 3% gelatin. Hespan, Ficoll-Paque or a two-step 3% gelatin followed by Ficoll-Paque separation. The two-step procedure was found to be superior to the other methods in enrichment of the Mk progenitor cells (CFU Mk) (34.3-fold), while at the same time retaining the number of myeloid and erythroid progenitors, CD34(+) and CD41(+) cells. In short-term (14d) liquid culture of non-adherent nucleated cells isolated by gelatin and Ficoll-Paque, a 40-fold expansion of clonable Mk progenitor cells was obtained in the presence of thrombopoietin (r-hu-TPO) and stem cell factor (r-hu-SCF). In similar cultures of isolated CD34(+) cells, a 100-fold clonable Mk progenitor was obtained at day 14. Therefore this new technique may facilitate the ex vivo expansion of Mk progenitor cells and be adopted for future use in CB transplantation.