Efficient retrovirus-mediated transduction of primitive human peripheral blood progenitor cells in stroma-free suspension culture
| Autor: | H Schrezenmeier, Patrizia Bossolasco, Davide Soligo, G. Lambertenghi Deliliers, Thomas Licht, K Schwarz, F Berger, B Kubanek |
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| Rok vydání: | 2001 |
| Předmět: |
DNA
Complementary medicine.medical_treatment Genetic enhancement Genetic Vectors Cell Culture Techniques Antigens CD34 Transduction (genetics) Retrovirus Stroma Transduction Genetic Genetics medicine Humans Rhodamine 123 Progenitor cell Growth Substances Molecular Biology P-glycoprotein biology Growth factor Cell Differentiation Genetic Therapy HLA-DR Antigens Hematopoietic Stem Cells biology.organism_classification Virology Cell biology Retroviridae biology.protein Cytokines Molecular Medicine Genes MDR Stem cell Cell Division |
| Zdroj: | Gene Therapy. 8:687-696 |
| ISSN: | 1476-5462 0969-7128 |
| Popis: | Retroviral transduction of hematopoietic cells has resulted in unsatisfactory gene marking in clinical studies. Since cytokine-stimulated stem cells have engrafted poorly in animal models, we investigated phenotypic changes during culture of peripheral blood progenitor cells (PBPC). Human CD34(+) HLA-DR(low) cells, immunomagnetically separated from PBPC collections, were found to extrude rhodamine-123, which is characteristic for primitive hematopoietic cells. Cells were grown in suspension cultures supplemented with cytokines. While interleukin-3-containing factor combinations promoted cell proliferation they caused loss of rhodamine-123 extrusion and reduced the frequencies of cobblestone area-forming cells (CAFC). Several other cytokines failed to stimulate cell divisions, which are required for retroviral transduction. A combination including Flt-3 ligand (FL), interleukin-6 and stem cell factor (SCF) preserved an immature phenotype for 5 to 6 days and stimulated cell divisions, which was improved upon addition of leukemia inhibitory factor and interleukin-11. Furthermore, the CAFC frequency among cells treated with these cytokines was increased as compared with widely used cocktails containing interleukin-3, interleukin-6 and SCF. Rhodamine-123 appeared to be a particularly sensitive indicator for differentiation of PBPC. For analysis of gene transfer, amphotropic retroviruses conferring an MDR1 cDNA were added repeatedly for 6 days to cytokine-treated PBPC stroma-free cultures. Proviral cDNA was detected by polymerase chain reaction in 68% of cobblestone areas derived from CD34(+)HLA-DR(low) cells that had been exposed to Flt-3 ligand, interleukin-6 and SCF. In summary, conditions were identified that facilitate efficient transduction of early PBPC with amphotropic retroviruses while preserving a primitive phenotype for extended periods. |
| Databáze: | OpenAIRE |
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