The high proliferative potential-quiescent (HPP-Q) cell assay allows an optimized evaluation of gene transfer efficiency into primitive hematopoieticstem/progenitor cells

Various protocols have been described to optimize gene transfer into hemato poietic cells. However, most of these methods do not specify whether they a re associated with an improved transduction of the more primitive stem/prog enitor cells, the best candidates for long-term engraftment. The majority o f these primitive cells remains in quiescence because of the negative contr ol of TGF-beta1, effective on these cells at low concentrations (10 pg/ml). in this study, CD34(+) cells were activated by a 10 h pretreatment with an ti-TGF-beta I followed by four successive retroviral supernatant incubation s of 6 h each. After 12 h (two incubations), a sig nificant increase in TGF -beta1 mRNA in CD34(+) cells was observed We wondered whether neo-synthesiz ed autocrine TGF-P I could induce reversion to quiescence of the more primi tive CD34(+) cells transduced after one cell cycle. This would prevent thei r subsequent detection in a classic clonal assay. Using the HPP-Q assay com paring a rapid mixed colony assay with or without anti-TGF-beta1, we indeed observed, that in clonal growth conditions the more primitive transduced c ells were activated and detectable only with anti-TGF-beta 1. Therefore, th is assay represents not only a rapid means to detect quiescent multipotent stem/progenitor cells but also a necessary step for the detection of the mo re primitive transduced cells which have returned to quiescence after retro viral induction of TGF-beta1 secretion.