title: Selective in vitro expansion and efficient retroviral transduction of human CD34+ CD38- haematopoietic stem cells.
abstract: Ex vivo expansion of primitive human haematopoietic stem cells (HSC) is clinically relevant for stem cell transplantation and gene therapy. Here, we demonstrate the selective expansion of CD34+CD38- cells from purified CD34+ cells upon stimulation with Flt3-ligand, stem cell factor and thrombopoietin. Over a 100-fold (range 80 to 128-fold) expansion of CD34+CD38- cells was observed with bone marrow and cord blood (CB). The expanded CD34+CD38- cells remained negative for lineage-specific markers and could be induced to differentiate into granulocytes, monocytes, megakaryocytes, erythrocytes, and T and B-lymphocytes in vitro. Lineage differentiation assays with single CD34+CD38- cells showed no loss of multilineage potential of expanded cells after ex vivo culture. We also demonstrated that the increase in frequency of CD34+CD38- cells was not as a result of the downregulation of CD38 expression during the culture. Quantitative analysis showed that the number of 6 week cobblestone area forming cells (CAFCwk6), a measure of proliferating HSC, in cytokine-stimulated CD34+ cells were increased by 20-fold. Expanded CD34+CD38- cells could be transduced efficiently with retroviruses encoding the low affinity nerve growth factor receptor (LNGFR) marker gene (17% to 44%, mean 27%), resulting in long-lasting expression of retroviral-encoded genes in progeny HSC and differentiated progenitors. We conclude that the combination Flt3-ligand (FL), stem cell factor and thrombopoietin (TPO) induced strong ex vivo proliferation of CD34+CD38- cells and that the absolute number of expanded cells with stem cell activity increased substantially in this population.
authors.affiliation: Departments of Hematology and Immunology, University Medical Center, Utrecht, The Netherlands.
authors.forename: Yuk Yin
authors.initials: YY
authors.lastname: Ng
authors.forename: Andries C
authors.initials: AC
authors.lastname: Bloem
authors.forename: Berris
authors.initials: B
authors.lastname: van Kessel
authors.forename: Henk
authors.initials: H
authors.lastname: Lokhorst
authors.forename: Ton
authors.initials: T
authors.lastname: Logtenberg
authors.forename: Frank J T
authors.initials: FJ
authors.lastname: Staal
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D018952:Antigens, CD34; D001854:Bone Marrow Cells / Q000276:immunology; D002452:Cell Count; D002454:Cell Differentiation; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D005312:Fetal Blood; D005822:Genetic Vectors / Q000008:administration & dosage; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D008565:Membrane Proteins / Q000494:pharmacology; D020800:Receptor, Nerve Growth Factor / Q000235:genetics; D019089:Stem Cell Factor / Q000494:pharmacology; D013268:Stimulation, Chemical; D013926:Thrombopoietin / Q000494:pharmacology; D014161:Transduction, Genetic
chemical_list: D018952:Antigens, CD34; D008565:Membrane Proteins; D020800:Receptor, Nerve Growth Factor; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D013926:Thrombopoietin