Cell cycle state, response to hemopoietic growth factors and retroviral vector-mediated transduction of human hemopoietic stem cells

A multi-parameter fluorometric analysis was applied to study in vitro proliferation and expansion of a candidate hemopoietic stem cell population, ie CD34brightLin cells. In primary hemopoietic cell samples the CD34brightLin population comprised on average, 1% of CD34+ cells from bone marrow (BM) or umbilical cord blood and 0.1% of mobilized peripheral blood CD34+ cells. The fraction of CD34brightLin cells engaged in the S+G2/M phases of the cell cycle was largest in regenerating BM (10.6% versus 1-2% in the other sources). Maintenance of CD34+ BM cells in hemopoietic growth factor supplemented liquid cultures resulted in a decrease of the CD34brightLin population in most samples. Cell cycle analysis showed the constant existence of proliferating CD34+ cells during the culture period. The fraction of cycling CD34brightLin cells was, however, very small and only occasionally exceeded input values. At all tested time-points during culture, BM CD34+ cells could be transduced by a single incubation with a recombinant retrovirus supernatant. CD34brightLin cells, however, were much more refractory to retroviral vector-mediated transduction. This could be explained only in part by quiescence of CD34brightLin cells. Hence, factors other than cell proliferation clearly influence the early stages of retroviral transduction of human hemopoietic stem cells