Immunophenotypic changes in cultured, primitive hematopoietic cells reflect the action of an unknown component in medium conditioned by derivatives of HT1080 cells rather than RD114-pseudotyped vector particles.

CD34⁺, CD38⁻ cells were isolated and placed in serum-free culture containing high-dose cytokines. After 24 hours of prestimulation, cells were transduced once at an MOI of 5 by various pseudotyped retroviral particles. Cells were then expanded for a total of 96 hours in culture and analyzed for phenotype. Under these conditions, untransduced cells maintained their phenotype (A). However, cells exposed to conditioned media derived from HT1080 cells and containing particles pseudotyped with either the RD114 envelope protein (B) or the amphotropic envelope protein (C) showed a significant shift to a CD38⁺ phenotype. Interestingly, no significant change in phenotype was noted in cells exposed to conditioned media from 3T3 cells containing amphotropic-pseudotyped vector particles (D) or RD114-pseudotyped particles derived transiently in 293T conditioned media (E). Cells exposed to RD114-pseudotyped retroviral particles derived from the HT1080 producer clone that had been preloaded on retronectin-coated dishes with removal of conditioned medium allowed efficient transduction without a change in the CD34⁺, CD38⁻ phenotype (F). Transduction based on EGFP expression was seen only in CD34⁺, CD38⁻ cells transduced with RD114/MGirL22Y vector particles (data not shown). These results suggest that the conditioned medium from the HT1080 cells and not the RD114-pseudotyped retroviral particles is responsible for the differentiation of the CD34⁺, CD38⁻cells.