Selection of a highly enriched population of retrovirus-infected human hematopoietic progenitor cells using SNL fibroblasts.
Retrovirus-mediated gene transfer into human hematopoietic progenitor cells for therapeutic or experimental purposes has proved difficult due to low and variable infection efficiency. To address this, we have developed an in vitro system for the selection and maintenance of a highly-enriched population of retrovirus-infected hematopoietic progenitor cells. Human umbilical cord CD34+ cells were cultured on SNL, a neo-containing murine fibroblast cell line used for embryonic stem cell culture. SNL-supported CD34+ cultures could be maintained with continuing blast cell and CFU-GM production for eight weeks, compared to four weeks in the absence of SNL. We then tested the ability of SNL to facilitate the selection in G418 of CD34+ cord cells infected with the neo-containing retrovirus, vsn-2. While all cells in the control cultures died within 14 days, vsn-2-infected CD34+ cells continued to proliferate, differentiate and produce CFU-GM for up to five weeks after infection. 100% of individually-plucked CFU-GM from such cultures were shown by PCR to be successfully infected. This approach should be useful for experimental work and, since it would diminish competitive repopulation between infected and uninfected progenitors, may also be utilized, with modification, for optimizing gene therapy protocols.
D016475:3T3 Cells; D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D001483:Base Sequence; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D003114:Colony-Forming Units Assay; D017931:DNA Primers; D005312:Fetal Blood; D005347:Fibroblasts / Q000166:cytology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D012190:Retroviridae / Q000235:genetics*; D013997:Time Factors; D014162:Transfection
D015703:Antigens, CD; D018952:Antigens, CD34; D017931:DNA Primers