title: Ex vivo expansion of early and late megakaryocyte progenitors.
abstract: Our goal is to produce ex vivo-expanded human megakaryocytes (MK) cells from peripheral blood progenitor cell (PBPC) harvests for use in supplementing conventional autografts. In this paper we show the megakaryocytopoietic productivity of small-scale in vitro serum-free cultures of human CD34+ cells containing MK growth and development factor (MGDF) and stem cell factor (Kit ligand; SCF) +/- granulocyte colony-stimulating factor (G-CSF). Cultures were characterized after 3, 6, 9, and 13 days by flow cytometry and clonogenic assays. CD34+ cells expanded 5.2- and 3.4-fold, and produced 2.2 and 2.4 CD34+/41(+) cells per seeded CD34+ cell after 6 and 9 days in culture, respectively. None were detected at day 13. CD41+ cells expanded exponentially over 13 days. Colony-forming unit-megakaryocyte (CFU-MK) also expanded exponentially, but the proportion of the most primitive CFU-MK dropped from 45% to 1.5% and to <1% after 6 and 9 days, respectively. G-CSF increased total cell expansion, but decreased CD41+ frequency, yielding no gain in MK production. We also found that PB CD34+ cells cultured for 3-6 days are richer in primitive MK progenitors, while those cultured for 9-13 days have greater numbers of more differentiated MKs. Overall, the combination of MGDF+SCF proved sufficient for expanding CD34+/CD41+ cells. As the stage of ex vivo MK differentiation most conducive to optimal platelet production in vivo is not known, we are planning a clinical trial to determine the efficacy of ex vivo-expanded MKs on platelet recovery in relation to MK maturity.
authors.affiliation: Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA.
authors.forename: P
authors.initials: P
authors.lastname: Lefebvre
authors.forename: J N
authors.initials: JN
authors.lastname: Winter
authors.forename: Y
authors.initials: Y
authors.lastname: Meng
authors.forename: I
authors.initials: I
authors.lastname: Cohen
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis; D001943:Breast Neoplasms / Q000473:pathology; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D016895:Culture Media, Serum-Free; D005260:Female; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology* / Q000276:immunology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology; D013997:Time Factors
chemical_list: D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor; D013926:Thrombopoietin