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 du e to low and variable infection efficiency. To address this, we have d eveloped an in vitro system for the selection and maintenance of a hig hly-enriched population or retrovirus-infected hematopoietic progenito r cells. Human umbilical cord CD34+ cells were cultured on SNL, a neo- containing murine fibroblast cell line used for embryonic stem cell cu lture. SNL-supported CD34+ cultures could be maintained with continuin g blast cell and CFU-GM production for eight weeks, compared to four w eeks in the absence of SNL. We then tested the ability of SNL to facil itate the selection in G418 of GD34+ 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 fa! prolifer ate, differentiate and produce CFU-GM for up to five weeks after infec tion. 100% of individually-plucked CFU-GM from such cultures were show n by PCR to be successfully infected. This approach should be useful f or experimental work and, since It would diminish competitive repopula tion between infected and uninfected progenitors, may also be utilized , with modification, for optimizing gene therapy protocols.