Retroviral Transduction of Human CD34+Umbilical Cord Blood Progenitor Cells with a Mutated Dihydrofolate Reductase cDNA
- 1 January 1998
- journal article
- research article
- Published by Mary Ann Liebert Inc in Human Gene Therapy
- Vol. 9 (1) , 63-71
- https://doi.org/10.1089/hum.1998.9.1-63
Abstract
Umbilical cord blood cells (UCB) have become a major target population for experimental and clinical studies using transfer of genes involved in inborn enzymatic diseases. Cord blood contains hematopoietic progenitor cells at a high frequency, and expanding these cells ex vivo generates sufficient numbers of hematopoietic precursors for transplantation into adults, e.g., as supportive treatment. As clinical reports about retroviral transduction into UCB cells have not been as encouraging as the first preclinical data, we have established a retroviral transduction system that allows expansion and selection of hematopoietic progenitor cells from UCB. CD34-enriched UCB cells were transduced with a retroviral vector encoding a mutated dihydrofolate reductase cDNA that confers MTX resistance. We observed increased resistance to MTX in transduced granulocyte macrophage-colony forming units (CFU-GM) after co-culture of CD34+ UCB cells with the virus-producing cell line, or after incubation with virus-containing supernatant. The supernatant-based transduction protocol included a prestimulation with recombinant interleukin-1 (rhIL-1), rhkit-ligand, and rhIL-3 to increase the percentage of cells in S phase to greater than 50%. Using this protocol we measured a 72-fold expansion of CFU-GM and a 2.5-fold selective advantage of transduced versus nontransduced progenitor cells after exposure to low-dose methotrexate in liquid culture. Polymerase chain reaction analysis revealed integration of proviral DNA into the majority of transduced colonies before and after ex vivo expansion. The retroviral vector and transduction protocol reported here provides an experimental system for selection and expansion of retrovirally transduced progenitor/stem cells from UCB that may help improve the efficiency of current clinical gene therapy strategies. UCB cells have a great potential for ex vivo expansion when cultured with appropriate cytokine combinations and can be used for transplantation after myeloablative therapy in children, and, possibly, in adults. They represent one possible source for pluripotent hematopoietic cells, and, therefore, are to be an interesting target for gene therapy trials. We have investigated the ex vivo expansion capacity of UCB progenitor cells with and without retroviral transduction and selection of successfully transduced cells. A retroviral vector containing a mutant DHFR gene conferring resistance to methotrexate (MTX) was introduced into CD34+ UCB cells. Progenitor cells could be extensively expanded in liquid culture and exposure to MTX in liquid culture resulted in a significant selection advantage of transduced progenitor cells.Keywords
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