Human cord blood CD34+CD38- cell transduction via lentivirus-based gene transfer vectors.
The efficient transfer and sustained expression of a transgene in human hematopoietic cells with in vivo repopulating potential would provide a significant advancement in the development of protocols for the treatment of hematopoietic diseases. Recent advances in the ability to purify and culture hematopoietic cells with the CD34+CD38- phenotype and with in vivo repopulating potential from human umbilical cord blood provide a direct means of testing the ability of transfer vectors to transduce these cells. Here we demonstrate the efficient transduction and expression of enhanced green fluorescent protein (EGFP) in human umbilical cord-derived CD34+CD38- cells, without prestimulation, using a lentivirus-based gene transfer system. Transduced CD34+CD38- cells cultured in serum-free medium supplemented with SCF, Flt-3, IL-3, and IL-6 maintained their surface phenotype for 5 days and expressed readily detectable levels of the transgene. The average transduction efficiency of the CD34+CD38- cells was 59 +/- 7% as determined by flow cytometry. Erythroid and myeloid colonies derived from transduced CD34+CD38- cells were EGFP positive at a high frequency (66 +/- 9%). In contrast, a murine leukemia virus-based vector transduced the CD34+CD38- cells at a low frequency (<4%). These results demonstrate the utility of lentiviral-based gene transfer vectors in the transduction of primitive human hematopoietic CD34+CD38- cells.
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
J T
JT
Evans
P F
PF
Kelly
E
E
O'Neill
J V
JV
Garcia
D000818:Animals; D018952:Antigens, CD34; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D005312:Fetal Blood; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006678:HIV; D006367:HeLa Cells; D006801:Humans; D016130:Immunophenotyping; D016086:Lentivirus; D009052:Leukemia Virus, Murine; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D019076:Transgenes; D014407:Tumor Cells, Cultured
D018952:Antigens, CD34; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins