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 Journal Article
| Authors: | Feugier, P.; Jo, D. Y.; Shieh, J.H.; Mackenzie, K. L.; Rafii, S.; Crystal, R. G.; Moore, M. A. S. |
| Article 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. |
| Keywords: | adult; controlled study; dna binding protein; human cell; genetics; dna-binding proteins; methodology; cytology; metabolism; cell division; cd34 antigen; stem cell factor; gene overexpression; bone marrow; membrane proteins; drug effect; pathology; cell population; vascularization; telomerase; gene vector; genetic transduction; genetic vectors; transduction, genetic; stem cell; endothelium cell; immunology; nonhodgkin lymphoma; genetic transfection; cell culture; umbilical vein; vascular endothelium; endothelium, vascular; umbilical veins; lymphoma, non-hodgkin; membrane protein; stem cell mobilization; hematopoietic stem cells; blood cell; blood cells; cell count; hematopoietic stem cell; flt3 ligand; granulocyte colony stimulating factor; antigens, cd34; genetic code; cell motility; hematopoietic stem cell mobilization; secretion; adenoviridae; biological factor; biological factors; coculture; coculture techniques; thrombopoietin; adenovirus; flt3 ligand protein; rna directed dna polymerase; humans; human; priority journal; article |
| Journal Title: | Journal of Hematotherapy and Stem Cell Research |
| Volume: | 11 |
| Issue: | 1 |
| ISSN: | 1525-8165 |
| Publisher: | Mary Ann Liebert, Inc |
| Date Published: | 2002-02-01 |
| Start Page: | 127 |
| End Page: | 138 |
| Language: | English |
| DOI: | 10.1089/152581602753448595 |
| PUBMED: | 11847009 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 14 November 2014 -- Source: Scopus |
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