title: Comparison of different methods for separation and ex vivo expansion of cord blood progenitor cells.
abstract: Umbilical cord blood is capable of hematopoietic stem cell reconstitution in children. However, the major limitation of cord blood is a relatively low content of pluripotent progenitor cells. Thus, safe engraftment for adolescents 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 gradient separation and a hydroxyethyl starch separation tended to produce a better 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 (median 78.35+/-7.1% vs. 67.2+/-5.5%). However, the recovery of CD34+ cells after magnetic cell sorting did not reach a statistically significant difference after the four different methods of red cell depletion, indicating that the recovery of WBC/MNC is not predictably correlated with the recovery of stem cells within these fractions. In a second step, we established three different cytokine combinations by adding the megakaryocyte growth and development factor +/- erythropoietin and granulocyte colony-stimulating factor to a fetal calf serum containing medium with Flt 3, stem cell factor, and interleukin-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-fold were obtained over a period of 7-14 days. These results demonstrate that cord blood stem cells can be expanded substantially in this short-term culture system.
authors.affiliation: Division of Pediatric Hematology and Oncology, Department of Pediatrics, University of Graz, Auenbruggerplatz 30, A-8036 Graz, Austria. wolfgang.schwinger@kfunigraz.ac.at
authors.forename: W
authors.initials: W
authors.lastname: Schwinger
authors.forename: M
authors.initials: M
authors.lastname: Benesch
authors.forename: H
authors.initials: H
authors.lastname: Lackner
authors.forename: R
authors.initials: R
authors.lastname: Kerbl
authors.forename: M
authors.initials: M
authors.lastname: Walcher
authors.forename: C
authors.initials: C
authors.lastname: Urban
mesh_terms: D018952:Antigens, CD34 / Q000097:blood; D001781:Blood Component Removal / Q000379:methods; D002469:Cell Separation / Q000379:methods*; D003114:Colony-Forming Units Assay; D016238:Cytapheresis; D017707:Erythrocyte Transfusion / Q000379:methods; D015672:Erythroid Precursor Cells / Q000166:cytology; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D006098:Granulocytes / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D008264:Macrophages / Q000166:cytology; D008533:Megakaryocytes / Q000166:cytology; D009000:Monocytes / Q000166:cytology; D011247:Pregnancy
chemical_list: D018952:Antigens, CD34