Comparison of different methods for separation and ex vivo expansion of cord blood progenitor cells

Umbilical cord blood is capable of hematopoietic stem cell reconstitution i n children. However, the major limitation of cord blood is a relatively low content of pluripotent progenitor cells. Thus, safe engraftment for adoles cents and for adults is still not predictable and a technology for ex vivo expansion of umbilical cord blood cells is desirable. In a first step, four different methods of red cell depletion followed by magnetic cell sorting of CD34+ cells were evaluated in this study in order to assess the efficacy and safety of optimal stem cell recovery. A modified two-step Ficoll gradi ent separation and a hydroxyethyl starch separation tended to produce a bet ter WBC/MNC recovery (median 94.2 +/- 2.44% vs. 90.2 +/- 5.8%) as compared with standard Ficoll gradient separation and a gelatin-based procedure (med ian 75.35 +/- 7.1% vs. 67.2 +/- 5.5%). However, the recovery of CD34+ cells after magnetic cell sorting did not reach a statistically significant diff erence after the four different methods of red cell depletion, indicating t hat the recovery of WBC/MNC is not predictably correlated with the recovery of stem cells within these fractions. In a second step, we established thr ee different cytokine combinations by adding the megakaryocyte growth and d evelopment factor +/- erythropoietin and granulocyte colony-stimulating fac tor to a fetal calf:serum containing medium with Fit 3, stem cell factor, a nd intrleukin-3. Net expansion of total colony-forming cells 20- to 50-fold and expansion of colony-forming cells after 5 weeks of culture 1.5- to 3-f old were obtained over a period of 7-14 days. These results demonstrate tha t cord blood stem cells can be expanded substantially in this short-term cu lture system.