CD34 stem/progenitor cells purified from cryopreserved normal cord blood can be transduced with high efficiency by a retroviral vector and expanded ex vivo with stable integration and expression of Fanconi anemia complementation C gene.
A future possibility for treatment of genetic diseases may be gene therapy using autologous cord blood (CB) stem/progenitor cells. This might require cryopreservation of CB stem/progenitor cells prior to purification, gene transduction, and ex vivo expansion of cells. To address this possibility, nonadherent low density T-lymphocyte depleted (NALT-) cells from fresh or cryopreserved cord blood were sorted for CD34 phenotype, transduced with a recombinant retroviral vector encoding Fanconi anemia complementation C (FACC) gene, and cells expanded ex vivo in suspension culture for 7 days with growth factors. The results demonstrate: 1) high recovery of viable cells after thawing; 2) high efficiency purification of CD34 cells from NALT- cells prior to and after cryopreservation; 3) high degree of expansion of nucleated cells and immature progenitors from CD34 cells before and after cryopreservation; 4) efficient transduction with stable integration and expression of newly introduced genes in cryopreserved and then sorted stem/progenitor cells, as detected prior to and after ex vivo expansion; and 5) high efficiency transduction of single isolated CD34 cells obtained from cryopreserved NALT- CB. This information should be of value for future studies evaluating the use of cryopreserved cord blood for gene transfer/gene therapy.
Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis 46202, USA.
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D018952:Antigens, CD34; D001483:Base Sequence; D018797:Cell Cycle Proteins; D002455:Cell Division; D002469:Cell Separation; D015925:Cryopreservation; D046508:Culture Techniques / Q000379:methods; D004268:DNA-Binding Proteins; D005199:Fanconi Anemia / Q000235:genetics*; D051856:Fanconi Anemia Complementation Group Proteins; D005312:Fetal Blood / Q000166:cytology* / Q000276:immunology; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics*; D012190:Retroviridae; D014161:Transduction, Genetic
D018952:Antigens, CD34; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051856:Fanconi Anemia Complementation Group Proteins; D009687:Nuclear Proteins; D011506:Proteins