Replication-deficient, recombinant adenovirus carrying the wild-type p53 gene (rAdp53) can significantly reduce tumor cell contamination of stem cell products from bre ist cancer patients. This provides a useful tumor cell purging strategy, which we have previous ly shown to have six log of activity. However, the safety of this procedure for self-renewii g, pluripotent hematopoietic stem cells has not been determined. The ability of human st(m cells to reconstitute hematopoiesis in mice with severe combined immunodeficiency (SCI D) and to undergo secondary transplantation provides a measure of self-renewing stem c ;11 activity. In these studies we examined the effect of rAd-p53 co-incubation with stem c;ll products on SCID «populating activity (SRA). At the targeted clinical dose (2 x I')10 particles/ml), rAd-p53 was co-cultured with mobilized human CD34 cells. The ability of these cells to establish multi-lineage, human-derived hematopoiesis in sublethally irradiat< :d, non-obese diabetic (NOD)-SCID mice was investigated. The presence of human cells w as determined by flow cytometry, in vitro granulocyte-macrophage colony-forming u lit assay and the demonstration of human Alu-sequences by PCR and Southern blottiiig. Limiting dilution analysis (LDA) provided a quantitative comparison between the SRA of control CD34 cells and CD34 cells co-incubated with rAd-p53. The LDA results in 1 le NOD-SCID mouse 6 weeks after transplantation were 1:94,170 (95% CI: 56,391 -157,256) and 1:50,747 (95% CI: 29,662 - 86,819) in the rAd-p53 co-incubated and control CD; 4' samples, respectively. Similar results were observed using flow cytometry data. Six wee ks after transplantation, marrow cells were harvested from the primary recipients and transplanted into secondary NOD-SCID mice. After 2 weeks, human cells were found in the marrow and spleen by flow cytometry. We conclude that the co-incubation of stem cell products with rAd-p53 is a safe purging protocol with little effect on self-renewiijg, pluripotent stem cells.
|Original language||English (US)|
|Issue number||11 PART I|
|State||Published - 2000|
ASJC Scopus subject areas
- Cell Biology