title: Ex vivo expansion of stem and progenitor cells in co-culture of mobilized peripheral blood CD34+ cells on human endothelium transfected with adenovectors expressing thrombopoietin, c-kit ligand, and Flt-3 ligand.
abstract: To optimize conditions for ex vivo expansion of adult hematopoietic stem cells, we evaluated the co-culture of G-CSF mobilized human peripheral blood (PB) CD34(+) cells with endothelial cells engineered to overexpress various hematopoietic growth factors. Immortalized human bone marrow endothelial cells (BMEC) transfected with an expression vector carrying cDNA encoding the human telomerase reverse transcriptase (hTERT) and human umbilical vein endothelial cells (HUVEC) were transfected with combinations of adenovectors expressing murine c-kit ligand (KL), human thrombopoietin (TPO), human Flt3 ligand (FL), and human granulocyte-macrophage colony-stimulating factor (GM-CSF). Ex vivo expansion of PB CD34(+) cells from normal donors and non-Hodgkin lymphoma (NHL) patients in endothelial co-culture was evaluated weekly for total cell production, progenitor (CFU-GM, BFU-E) cell production, and stem cell production as measured by Week-5 Cobblestone Area Forming Cell assay (Wk-5 CAFC). HUVEC transfected with adenovectors expressing TPO, KL, and FL provided the best co-culture system for expanding CD34(+) cells. Maximal total nuclear cell, CFU-GM, and Wk-5 CAFC production occurred between weeks 2 and 3 with 113-fold, 25-fold, and 2.2-5.5-fold expansions, respectively. We did not detect significant differences when GM-CSF was added to the co-culture system. Expansion was also obtained using recombinant human cytokines, but was not maintained beyond 3 weeks. We demonstrated that continuous generation of high levels of TPO, FL, and KL as well as other factors secreted by endothelium provided a clinically relevant co-culture method for ex vivo expansion of stem and progenitor cells from cryopreserved CD34(+) populations.
authors.affiliation: James Ewing Laboratory of Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 101, New York, NY 10021, USA. m-moore@ski.mskcc.org
authors.forename: Pierre
authors.initials: P
authors.lastname: Feugier
authors.forename: Deog Yeon
authors.initials: DY
authors.lastname: Jo
authors.forename: Jae Hung
authors.initials: JH
authors.lastname: Shieh
authors.forename: Karen L
authors.initials: KL
authors.lastname: MacKenzie
authors.forename: Shahin
authors.initials: S
authors.lastname: Rafii
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Crystal
authors.forename: Malcolm A S
authors.initials: MA
authors.lastname: Moore
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D018952:Antigens, CD34; D001685:Biological Factors / Q000235:genetics / Q000494:pharmacology*; D001773:Blood Cells / Q000166:cytology / Q000276:immunology; D001853:Bone Marrow / Q000098:blood supply; D002455:Cell Division / Q000187:drug effects; D018920:Coculture Techniques / Q000379:methods; D004268:DNA-Binding Proteins; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005822:Genetic Vectors; D019650:Hematopoietic Stem Cell Mobilization; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D008228:Lymphoma, Non-Hodgkin / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000494:pharmacology; D019089:Stem Cell Factor / Q000235:genetics / Q000494:pharmacology; D019098:Telomerase / Q000235:genetics; D013926:Thrombopoietin / Q000235:genetics / Q000494:pharmacology; D014161:Transduction, Genetic; D014471:Umbilical Veins
chemical_list: D018952:Antigens, CD34; D001685:Biological Factors; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D013926:Thrombopoietin; D019098:Telomerase